Targeted therapeutics

ABSTRACT

The present invention provides pharmacological compounds including an effector moiety conjugated to a binding moiety that directs the effector moiety to a biological target of interest. Likewise, the present invention provides compositions, kits, and methods (e.g., therapeutic, diagnostic, and imaging) including the compounds. The compounds can be described as a protein interacting binding moiety-drug conjugate (SDC-TRAP) compounds, which include a protein interacting binding moiety and an effector moiety. For example, in certain embodiments directed to treating cancer, the SDC-TRAP can include an Hsp90 inhibitor conjugated to a cytotoxic agent as the effector moiety.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 15/251,255, filed on Aug. 30, 2016, entitled “TARGETEDTHERAPEUTICS”, which is a continuation of PCT Application No.PCT/US2015/018442, entitled “TARGETED THERAPEUTICS”, filed on Mar. 3,2015, which claims the benefit of U.S. Provisional Patent ApplicationNo. 61/947,108, entitled, “TARGETED THERAPEUTICS”, filed on Mar. 3,2014, the entire contents of each of which are hereby incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to pharmacological compounds including aneffector moiety conjugated to a binding moiety that directs the effectormoiety to a biological target of interest. The compounds have broadpharmacological applications, including therapeutics, diagnostics, andimaging. For example, the compounds can specifically direct therapeuticeffector moieties to target cells or tissue of interest, for targetedchemotherapeutic treatment of conditions such as cancer.

BACKGROUND OF THE INVENTION

Although tremendous advances have been made in chemotherapy, currentlyavailable therapeutics and therapies remain unsatisfactory and theprognosis for the majority of patients diagnosed withchemotherapeutically treated diseases (e.g., cancer) remains poor.Often, the applicability and/or effectiveness of chemotherapy, as wellas other therapies and diagnostics employing potentially toxic moieties,is limited by undesired side effects.

Many disease and disorders are characterized by the presence of highlevels of certain proteins in specific types of cells. In some cases,the presence of these high levels of protein is caused byoverexpression. Historically, some of these proteins have been usefultargets for therapeutic molecules or used as biomarkers for thedetection of disease. One class of overexpressed intracellular proteinthat has been recognized as a useful therapeutic target is known as theheat shock proteins.

Heat shock proteins (HSPs) are a class of proteins that are up-regulatedin response to elevated temperature and other environmental stresses,such as ultraviolet light, nutrient deprivation, and oxygen deprivation.HSPs have many known functions, including acting as chaperones to othercellular proteins (called client proteins) to facilitate their properfolding and repair, and to aid in the refolding of misfolded clientproteins. There are several known families of HSPs, each having its ownset of client proteins. Hsp90 is one of the most abundant HSP families,accounting for about 1-2% of proteins in a cell that is not under stressand increasing to about 4-6% in a cell under stress.

Inhibition of Hsp90 results in degradation of its client proteins viathe ubiquitin proteasome pathway. Unlike other chaperone proteins, theclient proteins of Hsp90 are mostly protein kinases or transcriptionfactors involved in signal transduction, and a number of its clientproteins have been shown to be involved in the progression of cancer.Hsp90 has been shown by mutational analysis to be necessary for thesurvival of normal eukaryotic cells. However, Hsp90 is overexpressed inmany tumor types, indicating that it may play a significant role in thesurvival of cancer cells and that cancer cells may be more sensitive toinhibition of Hsp90 than normal cells. For example, cancer cellstypically have a large number of mutated and overexpressed oncoproteinsthat are dependent on Hsp90 for folding. In addition, because theenvironment of a tumor is typically hostile due to hypoxia, nutrientdeprivation, acidosis, etc., tumor cells may be especially dependent onHsp90 for survival. Moreover, inhibition of Hsp90 causes simultaneousinhibition of a number of oncoproteins, as well as hormone receptors andtranscription factors, making it an attractive target for an anti-canceragent. In view of the above, Hsp90 has been an attractive target of drugdevelopment, including such Hsp90 inhibitor (Hsp90i) compounds asganetespib, AUY-922, and IPI-504. At the same time, the advancement ofcertain of these compounds which showed early promise, e.g.,geldanamycin, has been slowed by those compounds' toxicity profile.Hsp90i compounds developed to date are believed to show great promise ascancer drugs, but other ways the ubiquity of Hsp90 in cancer cells mightbe leveraged have heretofore remained unexplored until now. Accordingly,the need exists for therapeutic molecules that selectively targetproteins, such as Hsp90, that are overexpressed in cells associated withparticular diseases or disorders.

SUMMARY OF THE INVENTION

The present invention provides pharmacological molecules (“SDC-TRAPs”)including an effector moiety conjugated to a binding moiety, whichdirects the effector moiety into a target cell of interest in a mannerthat traps the molecule in the target cell. In a specific embodiment,the effector moiety is conjugated via a cleavable bond or linker to thebinding moiety, such that the cleavable bond or linker is preferentiallycleaved after the SDC-TRAP enters the target cell. The inventors of theinstant application have discovered that the SDC-TRAP molecules of theinvention can be used to selectively deliver an effector moiety to aspecific type of cell in order to increase the intracellular level ofthe effector moiety in the target cell as compared to other cells. Theinventors have demonstrated that certain SDC-TRAP molecules of theinvention enter target cells by passive diffusion and are selectivelyretained in the target cells. Specifically, the inventors have shownthat certain SDC-TRAP molecules of the invention are selectivelyretained only in cells that overexpress or otherwise have a highintracellular level of the protein to which the binding moiety binds.There are numerous advantages to these SDC-TRAP molecules and to methodsof using these molecules that are described herein.

Specifically, the invention provides SDC-TRAP molecules that aretargeted to cells of interest and trapped intracellularly for asufficient period of time such that the effector moiety has the desiredbiological effect. In one embodiment, these SDC-TRAPs allow for thetargeting of an effector moiety to a particular type of cell based onthe overexpression of an intracellular protein that is characteristic ofa particular disease or disorder. Accordingly, the present inventionprovides compositions, kits, and methods (e.g., therapeutic, diagnostic,and imaging) including the compounds.

In a specific embodiment, the application exemplifies the use of Hsp90interacting moieties, e.g., inhibitors, as the binding moiety in theSDC-TRAPs. However, the invention is intended to include other bindingmoieties, including those that are contemplated, listed and exemplifiedherein. Accordingly, in certain embodiments directed to treating canceror inflammation, the SDC-TRAP includes an Hsp90 inhibitor moietyconjugated to an effector moiety. In certain embodiments, the effectormoiety is a cytotoxic effector moiety.

In another embodiment, the SDC-TRAP includes an effector moiety that iseffective while still linked to the binding moiety. In such embodiment,cleavage of the bond or linker in the target cell is not a necessaryfeature of the invention. In other cases, such as cytotoxic effectormoieties, the effector moiety should only be effective after the linkeror bond is cleaved and the effector moiety is released from the SDC-TRAPmolecule inside the target cell. In either case, SDC-TRAPs that do notenter into the target cell should be rapidly cleared (e.g., from theplasma or other non-target cells or tissues).

In another embodiment, the binding moiety of the SDC-TRAP binds aprotein within the target cell, which may itself produce a desiredbiological effect (e.g., such as inhibiting Hsp90 within the targetcell). In one embodiment, the binding moiety can contribute to theoverall efficacy of the SDC-TRAP by not only binding an intracellularprotein present in the target cell but by also conveying a particulardesired biological effect. For example, if the binding moiety is anHsp90 inhibitor and the target cell is a cancer cell, than the overallactivity of the SDC-TRAP may not only result from the effector moiety,but also from the biological activity of the Hsp90 inhibitor.

Alternatively, interaction of the binding moiety with its protein targetmay not impart a biological effect, but rather only serve to attract andretain the SDC-TRAP within the target cell. In this embodiment, thebinding moiety may reversibly bind to the intracellular target proteinand create an intracellular equilibrium between free and bound SDC-TRAPmolecules. This equilibrium may allow for cleavage of the SDC-TRAP andmore effective delivery of the effector moiety, e.g., release of theeffector moiety from the binding moiety by, for example, enzymaticcleavage, hydrolysis or degradation. In some cases, the effector moietymay be inactive until such release occurs.

In various aspects and embodiments, the present invention providesnumerous advantages. For example, the SDC-TRAP can provide for targetedtherapy, maximizing efficacy and/or minimizing undesired side effects.The SDC-TRAP can provide for targeted use of an effector moiety thatwould otherwise be unsuitable for administration alone due to toxicityand/or undesired systemic effects. The SDC-TRAP can facilitate targetingsuch effector moieties to intracellular targets—that is, due to its sizeand chemical properties, the SDC-TRAP can passively diffuse (or in somecases be actively transported) into a cell having an intracellulartarget of interest. Alternatively, the SDC-TRAP can deliver in aselective manner a cytotoxic molecule to destroy a target cell, such asa cancer or inflammatory cell.

Additional advantages are discussed in detail below.

These and other advantages of the present invention are of particularinterest, for example, in chemotherapy where despite tremendous recentadvances, currently available therapeutics and therapies remainsunsatisfactory and the prognosis for the majority of patients diagnosedwith diseases such as cancer remains poor. However, while many of theillustrative embodiments and examples are presented in the context ofcancer, a person of ordinary skill in the art would understand that thepresent invention has applications across therapeutic, diagnostic, andimaging applications that require, or would benefit from, targeting ofan effector moiety.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety and an effector moiety.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety and an effector moiety, wherein the SDC-TRAP is able toenter a cell by active transport.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety and an effector moiety, wherein the SDC-TRAP has amolecular weight of less than about 1600 Daltons.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety and an effector moiety, wherein the SDC-TRAP has amolecular weight of less than about 1200 Daltons.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety and an effector moiety, wherein the SDC-TRAP has amolecular weight of less than about 800 Daltons.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety and an effector moiety, wherein the SDC-TRAP has amolecular weight of less than about 600 Daltons.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety and an effector moiety, wherein the SDC-TRAP has amolecular weight of less than about 400 Daltons.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety and an effector moiety, wherein the binding moiety has amolecular weight of less than about 800 Daltons.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety and an effector moiety, wherein the effector moiety has amolecular weight of less than 800 Daltons.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety and an effector moiety, wherein the binding moiety andthe effector moiety are approximately equal in size.

In various aspects, the invention provides an SDC-TRAP comprising anHsp90 binding moiety and an effector moiety, wherein the Hsp90 bindingmoiety interacts with the N-terminal domain of Hsp90.

In various aspects, the invention provides an SDC-TRAP comprising anHsp90 binding moiety and an effector moiety, wherein the Hsp90 bindingmoiety interacts with the C-terminal domain of Hsp90.

In various aspects, the invention provides an SDC-TRAP comprising anHsp90 binding moiety and an effector moiety, wherein the Hsp90 bindingmoiety interacts with the middle domain of Hsp90.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety and an effector moiety, wherein the binding moietyinteracts with a predetermined domain of a multidomain target proteinmolecule.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety (e.g., an Hsp90 binding moiety) and an effector moiety,wherein the binding moiety (e.g., Hsp90 binding moiety) has a K_(d) of100 nM or higher (e.g., for a predetermined target molecule, forexample, Hsp90).

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety (e.g., Hsp90 binding moiety) and an effector moiety,wherein when administered to a subject, the SDC-TRAP is present at aratio of 2:1 in target (e.g., tumor) cells compared to plasma. Inanother embodiment, the invention provides an SDC-TRAP comprising abinding moiety (e.g., Hsp90 binding moiety) and an effector moiety,wherein when administered to a subject the SDC-TRAP present at a ratioof 2:1 in target (e.g., tumor) cells compared to normal cells.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety (e.g., Hsp90 binding moiety) and an effector moiety,wherein the SDC-TRAP is present in target (e.g., cancer) cells for atleast 24 hours.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety (e.g., Hsp90 binding moiety) and an effector moiety,wherein the effector moiety is released for a period of at least 6 hours(e.g., within a target cell and/or tissue).

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety (e.g., Hsp90 binding moiety) and an effector moiety,wherein the effector moiety is selectively released inside a target(e.g., cancer) cell.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety (e.g., Hsp90 binding moiety) and an effector moiety,wherein the SDC-TRAP allows for the use of an effector moiety that istoxic or otherwise unfit for administration to a subject.

In various aspects, the invention provides an SDC-TRAP comprising abinding moiety (e.g., Hsp90 binding moiety) and an effector moiety,wherein the Hsp90 is an inhibitor (e.g., Hsp90 inhibitor) that isineffective as a therapeutic agent when administered alone.

In various aspects, the invention provides an SDC-TRAP comprising anHsp90 binding moiety and an effector moiety.

In various aspects, the invention provides pharmaceutical compositionscomprising a therapeutically effective amount of at least one SDC-TRAP,and at least one pharmaceutical excipient.

In various aspects, the invention provides methods for treating asubject in need thereof comprising administering a therapeuticallyeffective amount of at least one SDC-TRAP to the subject, therebytreating the subject.

In various aspects, the invention provides methods for imaging,diagnosing, and/or selecting a subject comprising administering aneffective amount of at least one SDC-TRAP to the subject, therebyimaging, diagnosing, and/or selecting the subject.

In various aspects, the invention provides kits for treating a subjectin need thereof comprising at least one SDC-TRAP and instruction foradministering a therapeutically effective amount of the at least oneSDC-TRAP to the subject, thereby treating the subject.

In various aspects, the invention provides kits for imaging, diagnosing,and/or selecting a subject comprising at least one SDC-TRAP andinstruction for administering an effective amount of at least oneSDC-TRAP to the subject, thereby imaging, diagnosing, and/or selectingthe subject.

In various embodiments, the invention can include any one or more of theaspects disclosed herein having any one or more of the featuresdisclosed herein.

In various embodiments, the binding moiety interacts with a protein thatis overexpressed in cancerous cells compared to normal cells.

In various embodiments, the protein is a chaperonin protein. Thechaperonin can be, for example, Hsp90.

In various embodiments, the chaperonin is an Hsp90 binding moiety.

In various embodiments, the binding moiety is an Hsp90 ligand or aprodrug thereof. The Hsp90 ligand can be, for example, an Hsp90inhibitor. An Hsp90 inhibitor can be selected from the group consistingof geldanamycins, macbecins, tripterins, tanespimycins, and radicicols.

In various embodiments, the binding moiety can be an Hsp90-targetingmoiety, for example a triazole/resorcinol-based compound that bindsHsp90, or a resorcinol amide-based compound that binds Hsp90, e.g.,ganetespib, AUY-922, or AT-13387.

In various embodiments, the binding moiety can be an Hsp90-bindingcompound of formula (I):

whereinR¹ may be alkyl, aryl, halide, carboxamide or sulfonamide; R² may bealkyl, cycloalkyl, aryl or heteroaryl, wherein when R² is a six-memberedaryl or heteroaryl, R² is substituted at the 3- and 4-positions relativeto the connection point on the triazole ring, through which a linker Lis attached; and R³ may be SH, OH, —CONHR⁴, aryl or heteroaryl, whereinwhen R³ is a six-membered aryl or heteroaryl, R³ is substituted at the 3or 4 position.

In various embodiments, the binding moiety can be an Hsp90-bindingcompound of formula (II):

whereinR¹ may be alkyl, aryl, halo, carboxamido, sulfonamido; and R² may beoptionally substituted alkyl, cycloalkyl, aryl or heteroaryl. Examplesof such compounds include5-(2,4-dihydroxy-5-isopropylphenyl)-N-(2-morpholinoethyl)-4-(4-(morpholinomethyl)phenyl)-4H-1,2,4-triazole-3-carboxamideand5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(4-methylpiperazin-1-yl)phenyl)-N-(2,2,2-trifluoroethyl)-4H-1,2,4-triazole-3-carboxamide.

In various embodiments, the binding moiety can be an Hsp90-bindingcompound of formula (III):

whereinX, Y, and Z may independently be CH, N, O or S (with appropriatesubstitutions and satisfying the valency of the corresponding atoms andaromaticity of the ring); R¹ may be alkyl, aryl, halide, carboxamido orsulfonamido; R² may be substituted alkyl, cycloalkyl, aryl orheteroaryl, where a linker L is connected directly or to the extendedsubstitutions on these rings; R³ may be SH, OH, NR⁴R⁵ AND —CONHR⁶, towhich an effector moiety may be connected; R⁴ and R⁵ may independentlybe H, alkyl, aryl, or heteroaryl; and R⁶ may be alkyl, aryl, orheteroaryl, having a minimum of one functional group to which aneffector moiety may be connected.

As used herein, the term “alkyl” means a saturated straight chain orbranched non-cyclic hydrocarbon having from 1 to 10 carbon atoms.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl andn-decyl; while saturated branched alkyls include isopropyl, sec-butyl,isobutyl, tert-butyl, isopentyl, 2-methylbutyl, 3-methylbutyl,2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-methylhexyl,3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylbutyl,2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,3-dimethylhexyl,2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylpentyl,2,2-dimethylhexyl, 3,3-dimtheylpentyl, 3,3-dimethylhexyl,4,4-dimethylhexyl, 2-ethylpentyl, 3-ethylpentyl, 2-ethylhexyl,3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl,2-methyl-3-ethylpentyl, 2-methyl-4-ethylpentyl, 2-methyl-2-ethylhexyl,2-methyl-3-ethylhexyl, 2-methyl-4-ethylhexyl, 2,2-diethylpentyl,3,3-diethylhexyl, 2,2-diethylhexyl, 3,3-diethylhexyl and the like. Theterm “(C₁-C₆)alkyl” means a saturated straight chain or branchednon-cyclic hydrocarbon having from 1 to 6 carbon atoms. Representative(C₁-C₆)alkyl groups are those shown above having from 1 to 6 carbonatoms. Alkyl groups included in compounds of this invention may beoptionally substituted with one or more substituents.

As used herein, the term “alkenyl” means a saturated straight chain orbranched non-cyclic hydrocarbon having from 2 to 10 carbon atoms andhaving at least one carbon-carbon double bond. Representative straightchain and branched (C₂-C₁₀)alkenyls include vinyl, allyl, 1-butenyl,2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl,2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, 1-hexenyl, 2-hexenyl,3-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 1-octenyl, 2-octenyl,3-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 2-decenyl,3-decenyl and the like. Alkenyl groups may be optionally substitutedwith one or more substituents.

As used herein, the term “alkynyl” means a saturated straight chain orbranched non-cyclic hydrocarbon having from 2 to 10 carbon atoms andhaving at least one carbon-carbon triple bond. Representative straightchain and branched alkynyls include acetylenyl, propynyl, 1-butynyl,2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, 4-pentynyl,1-hexynyl, 2-hexynyl, 5-hexynyl, 1-heptynyl, 2-heptynyl, 6-heptynyl,1-octynyl, 2-octynyl, 7-octynyl, 1-nonynyl, 2-nonynyl, 8-nonynyl,1-decynyl, 2-decynyl, 9-decynyl, and the like. Alkynyl groups may beoptionally substituted with one or more substituents.

As used herein, the term “cycloalkyl” means a saturated, mono- orpolycyclic alkyl radical having from 3 to 20 carbon atoms.Representative cycloalkyls include cyclopropyl, 1-methylcyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,cyclononyl, -cyclodecyl, octahydro-pentalenyl, and the like. Cycloalkylgroups may be optionally substituted with one or more substituents.

As used herein, the term “cycloalkenyl” means a mono- or poly-cyclicnon-aromatic alkyl radical having at least one carbon-carbon double bondin the cyclic system and from 3 to 20 carbon atoms. Representativecycloalkenyls include cyclopentenyl, cyclopentadienyl, cyclohexenyl,cyclohexadienyl, cycloheptenyl, cycloheptadienyl, cycloheptatrienyl,cyclooctenyl, cyclooctadienyl, cyclooctatrienyl, cyclooctatetraenyl,cyclononenyl, cyclononadienyl, cyclodecenyl, cyclodecadienyl,1,2,3,4,5,8-hexahydronaphthalenyl and the like. Cycloalkenyl groups maybe optionally substituted with one or more substituents.

As used herein, the term “haloalkyl” means and alkyl group in which oneor more (including all) the hydrogen radicals are replaced by a halogroup, wherein each halo group is independently selected from —F, —Cl,—Br, and —I. The term “halomethyl” means a methyl in which one to threehydrogen radical(s) have been replaced by a halo group. Representativehaloalkyl groups include trifluoromethyl, bromomethyl,1,2-dichloroethyl, 4-iodobutyl, 2-fluoropentyl, and the like.

As used herein, an “alkoxy” is an alkyl group which is attached toanother moiety via an oxygen linker.

As used herein, an “haloalkoxy” is an haloalkyl group which is attachedto another moiety via an oxygen linker.

As used herein, the term an “aromatic ring” or “aryl” means ahydrocarbon monocyclic or polycyclic radical in which at least one ringis aromatic. Examples of suitable aryl groups include, but are notlimited to, phenyl, tolyl, anthracenyl, fluorenyl, indenyl, azulenyl,and naphthyl, as well as benzo-fused carbocyclic moieties such as5,6,7,8-tetrahydronaphthyl. Aryl groups may be optionally substitutedwith one or more substituents. In one embodiment, the aryl group is amonocyclic ring, wherein the ring comprises 6 carbon atoms, referred toherein as “(C₆)aryl.”

As used herein, the term “aralkyl” means an aryl group that is attachedto another group by a (C₁-C₆)alkylene group. Representative aralkylgroups include benzyl, 2-phenyl-ethyl, naphth-3-yl-methyl and the like.Aralkyl groups may be optionally substituted with one or moresubstituents.

As used herein, the term “alkylene” refers to an alkyl group that hastwo points of attachment. The term “(C₁-C₆)alkylene” refers to analkylene group that has from one to six carbon atoms. Straight chain(C₁-C₆)alkylene groups are preferred. Non-limiting examples of alkylenegroups include methylene (—CH₂—), ethylene (—CH₂CH₂—), n-propylene(—CH₂CH₂CH₂—), isopropylene (—CH₂CH(CH₃)—), and the like. Alkylenegroups may be optionally substituted with one or more substituents.

As used herein, the term “heterocyclyl” means a monocyclic (typicallyhaving 3- to 10-members) or a polycyclic (typically having 7- to20-members) heterocyclic ring system which is either a saturated ring ora unsaturated non-aromatic ring. A 3- to 10-membered heterocycle cancontain up to 5 heteroatoms; and a 7- to 20-membered heterocycle cancontain up to 7 heteroatoms. Typically, a heterocycle has at least oncarbon atom ring member. Each heteroatom is independently selected fromnitrogen, which can be oxidized (e.g., N(O)) or quaternized; oxygen; andsulfur, including sulfoxide and sulfone. The heterocycle may be attachedvia any heteroatom or carbon atom. Representative heterocycles includemorpholinyl, thiomorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl,piperazinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl,tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyrindinyl,tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, andthe like. A heteroatom may be substituted with a protecting group knownto those of ordinary skill in the art, for example, the hydrogen on anitrogen may be substituted with a tert-butoxycarbonyl group.Furthermore, the heterocyclyl may be optionally substituted with one ormore substituents. Only stable isomers of such substituted heterocyclicgroups are contemplated in this definition.

As used herein, the term “heteroaromatic”, “heteroaryl” or like termsmeans a monocyclic or polycyclic heteroaromatic ring comprising carbonatom ring members and one or more heteroatom ring members. Eachheteroatom is independently selected from nitrogen, which can beoxidized (e.g., N(O)) or quaternized; oxygen; and sulfur, includingsulfoxide and sulfone. Representative heteroaryl groups include pyridyl,1-oxo-pyridyl, furanyl, benzo[1,3]dioxolyl, benzo[1,4]dioxinyl, thienyl,pyrrolyl, oxazolyl, imidazolyl, thiazolyl, a isoxazolyl, quinolinyl,pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, atriazinyl, triazolyl, thiadiazolyl, isoquinolinyl, indazolyl,benzoxazolyl, benzofuryl, indolizinyl, imidazopyridyl, tetrazolyl,benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzoxadiazolyl,indolyl, tetrahydroindolyl, azaindolyl, imidazopyridyl, quinazolinyl,purinyl, pyrrolo[2,3]pyrimidinyl, pyrazolo[3,4]pyrimidinyl,imidazo[1,2-a]pyridyl, and benzothienyl. In one embodiment, theheteroaromatic ring is selected from 5-8 membered monocyclic heteroarylrings. The point of attachment of a heteroaromatic or heteroaryl ring toanother group may be at either a carbon atom or a heteroatom of theheteroaromatic or heteroaryl rings. Heteroaryl groups may be optionallysubstituted with one or more substituents.

As used herein, the term “(C₅)heteroaryl” means an aromatic heterocyclicring of 5 members, wherein at least one carbon atom of the ring isreplaced with a heteroatom such as, for example, oxygen, sulfur ornitrogen. Representative (C₅)heteroaryls include furanyl, thienyl,pyrrolyl, oxazolyl, imidazolyl, thiazolyl, isoxazolyl, pyrazolyl,isothiazolyl, pyrazinyl, triazolyl, thiadiazolyl, and the like.

As used herein, the term “(C₆)heteroaryl” means an aromatic heterocyclicring of 6 members, wherein at least one carbon atom of the ring isreplaced with a heteroatom such as, for example, oxygen, nitrogen orsulfur. Representative (C₆)heteroaryls include pyridyl, pyridazinyl,pyrazinyl, triazinyl, tetrazinyl and the like.

As used herein, the term “heteroaralkyl” means a heteroaryl group thatis attached to another group by a (C₁-C₆)alkylene. Representativeheteroaralkyls include 2-(pyridin-4-yl)-propyl, 2-(thien-3-yl)-ethyl,imidazol-4-yl-methyl and the like. Heteroaralkyl groups may beoptionally substituted with one or more substituents.

As used herein, the term “halogen” or “halo” means —F, —Cl, —Br or —I.

Suitable substituents for an alkyl, alkylene, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, heterocyclyl, aryl, aralkyl, heteroaryl, andheteroaralkyl groups include any substituent which will form a stablecompound of the invention. Examples of substituents for an alkyl,alkylene, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl,aryl, aralkyl, heteroaryl, and heteroarylalkyl include an optionallysubstituted alkyl, an optionally substituted alkenyl, an optionallysubstituted alkynyl, an optionally substituted cycloalkyl, an optionallysubstituted cycloalkenyl, an optionally substituted heterocyclyl, anoptionally substituted aryl, an optionally substituted heteroaryl, anoptionally substituted aralkyl, an optionally substituted heteraralkyl,or a haloalkyl.

In addition, alkyl, cycloalkyl, alkylene, a heterocyclyl, and anysaturated portion of a alkenyl, cycloalkenyl, alkynyl, aralkyl, andheteroaralkyl groups, may also be substituted with ═O, or ═S.

When a heterocyclyl, heteroaryl, or heteroaralkyl group contains anitrogen atom, it may be substituted or unsubstituted. When a nitrogenatom in the aromatic ring of a heteroaryl group has a substituent thenitrogen may be a quaternary nitrogen.

As used herein, the term “lower” refers to a group having up to fouratoms. For example, a “lower alkyl” refers to an alkyl radical havingfrom 1 to 4 carbon atoms, “lower alkoxy” refers to “—O—(C₁-C₄)alkyl anda “lower alkenyl” or “lower alkynyl” refers to an alkenyl or alkynylradical having from 2 to 4 carbon atoms, respectively.

Unless indicated otherwise, the compounds of the invention containingreactive functional groups (such as (without limitation) carboxy,hydroxy, thiol, and amino moieties) also include protected derivativesthereof. “Protected derivatives” are those compounds in which a reactivesite or sites are blocked with one or more protecting groups. Examplesof suitable protecting groups for hydroxyl groups include benzyl,methoxymethyl, allyl, trimethylsilyl, tert-butyldimethylsilyl, acetate,and the like. Examples of suitable amine protecting groups includebenzyloxycarbonyl, tert-butoxycarbonyl, tert-butyl, benzyl andfluorenylmethyloxy-carbonyl (Fmoc). Examples of suitable thiolprotecting groups include benzyl, tert-butyl, acetyl, methoxymethyl andthe like. Other suitable protecting groups are well known to those ofordinary skill in the art and include those found in T. W. Greene,Protecting Groups in Organic Synthesis, John Wiley & Sons, Inc. 1981.

Exemplary Hsp90 inhibitors include those disclosed in U.S. Pat. Nos.8,362,055 and 7,825,148. Examples of such compounds include

In various embodiments, the binding moiety can be an Hsp90-bindingcompound of formula (IV):

whereinR¹ may be alkyl, aryl, halo, carboxamido or sulfonamido; R² and R³ areindependently C₁-C₅ hydrocarbyl groups optionally substituted with oneor more of hydroxy, halogen, C₁-C₂ alkoxy, amino, mono- and di-C₁-C₂alkylamino; 5- to 12-membered aryl or heteroaryl groups; or, R² and R³,taken together with the nitrogen atom to which they are attached, form a4- to 8-membered monocyclic heterocyclic group, of which up to 5 ringmembers are selected from O, N and S.

Examples of such compounds include

In various embodiments, the binding moiety includes an Hsp90-targetingmoiety, for example one or more geldanamycins, e.g.,

macbecins, tripterins, tanespimycins, e.g.,

novobiocin (a C-terminal Hsp90i.)

In various embodiments, the effector moiety is a therapeutic moiety. Thetherapeutic moiety can be, for example, a cytotoxic moiety. A cytotoxicmoiety can be SN-38, bendamustine, a vascular disrupting agent (VDA),doxorubicin, pemetrexed, vorinostat, lenalidomide, irinotecan,ganetespib, docetaxel, 17-AAG, 5-FU, abiraterone, crizotinib, KW-2189,BUMB2, DC1, CC-1065, adozelesin, fulvestrant, topotecan or (a)fragment(s) thereof.

In various embodiments, the effector moiety may include pan-CDKInhibitors, such as flavopiridol; EGFR/EGFR2 inhibitors, such aslapatinib; VEGFR inhibitors, such as axitinib; mBRAF inhibitors, such asvemurafenib; BCR-ABL/Kit inhibitors, such as imatinib; multi-targetkinase inhibitors, such as staurosporine; epigenetic regulators, such aspanobinostat; proteasome inhibitors, such as carfilzomib; and IDOinhibitors, such as INCB024360 and methyl tryptophan.

In various embodiments, the effector moiety is an antifolate orfragments thereof (e.g., temozolamide, mitozolamide, nitrogen mustards,estramustine, or chloromethine).

In various embodiments, the effector moiety includes one or more:peptidyl-prolyl isomerase ligands, e.g., FK506 (tacrolimus); rapamycin,cyclosporin A; steroid hormone receptor ligands, e.g., naturallyoccurring steroid hormones, such as estrogen, progestin, testosterone,as well as synthetic derivatives and mimetics thereof; small moleculesthat bind to cytoskeletal proteins, e.g., antimitotic agents, such astaxanes, colchicine, colcemid, nocadozole, vinblastine, and vincristine,actin binding agents, such as cytochalasin, latrunculin, phalloidin;lenalidomide, pomalidomide, camptothecins including

topotecan, combretastatins, capecitabine, gemcitabine, vinca alkaloids,platinum-containing compounds, metformin, HDAC inhibitors (e.g.,suberoylanilidehydroxamic acid (SAHA)), thymidylate synthase inhibitorssuch as methotrexate, pemetrexed, and raltitrexed; nitrogen mustardssuch as bendamustine and melphalan; 5-fluorouracil (5-FU) and itsderivatives; and agents used in ADC drugs, such as vedotin and DM1.

In various embodiments, the effector moiety is derived from one or more:central nervous system depressants, e.g., general anesthetics(barbiturates, benzodiazepines, steroids, cyclohexanone derivatives, andmiscellaneous agents), sedative-hypnotics (benzodiazepines,barbiturates, piperidinediones and triones, quinazoline derivatives,carbamates, aldehydes and derivatives, amides, acyclic ureides,benzazepines and related drugs, phenothiazines), central voluntarymuscle tone modifying drugs (anticonvulsants, such as hydantoins,barbiturates, oxazolidinediones, succinimides, acylureides,glutarimides, benzodiazepines, secondary and tertiary alcohols,dibenzazepine derivatives, valproic acid and derivatives, GABA analogs),analgesics (morphine and derivatives, oripavine derivatives, morphinanderivatives, phenylpiperidines, 2,6-methane-3-benzazocaine derivatives,diphenylpropylamines and isosteres, salicylates, p-aminophenolderivatives, 5-pyrazolone derivatives, arylacetic acid derivatives,fenamates and isosteres) and antiemetics (anticholinergics,antihistamines, antidopaminergics); central nervous system stimulants,e.g., analeptics (respiratory stimulants, convulsant stimulants,psychomotor stimulants), narcotic antagonists (morphine derivatives,oripavine derivatives, 2,6-methane-3-benzoxacine derivatives, morphinanderivatives) nootropics; psychopharmacological/psychotropics, e.g.,anxiolytic sedatives (benzodiazepines, propanediol carbamates)antipsychotics (phenothiazine derivatives, thioxanthine derivatives,other tricyclic compounds, butyrophenone derivatives and isosteres,diphenylbutylamine derivatives, substituted benzamides, arylpiperazinederivatives, indole derivatives), antidepressants (tricyclic compounds,MAO inhibitors).

In various embodiments, the effector moiety is derived from one or more:respiratory tract drugs, e.g., central antitussives (opium alkaloids andtheir derivatives); immunosuppressive agents; pharmacodynamic agents,such as peripheral nervous system drugs, e.g., local anesthetics (esterderivatives, amide derivatives); drugs acting at synaptic orneuroeffector junctional sites, e.g., cholinergic agents, cholinergicblocking agents, neuromuscular blocking agents, adrenergic agents,antiadrenergic agents; smooth muscle active drugs, e.g., spasmolytics(anticholinergics, musculotropic spasmolytics), vasodilators, smoothmuscle stimulants; histamines and antihistamines, e.g., histamine andderivative thereof (betazole), antihistamines (H₁-antagonists,H₂-antagonists), histamine metabolism drugs; cardiovascular drugs, e.g.,cardiotonics (plant extracts, butenolides, pentadienolids, alkaloidsfrom erythrophleum species, ionophores,-adrenoceptor stimulants),antiarrhythmic drugs, antihypertensive agents, antilipidemic agents(clofibric acid derivatives, nicotinic acid derivatives, hormones andanalogs, antibiotics, salicylic acid and derivatives), antivaricosedrugs, hemostyptics; chemotherapeutic agents, such as anti-infectiveagents, e.g., ectoparasiticides (chlorinated hydrocarbons, pyrethins,sulfurated compounds), anthelmintics, antiprotozoal agents, antimalarialagents, antiamebic agents, antileiscmanial drugs, antitrichomonalagents, antitrypanosomal agents, sulfonamides, antimycobacterial drugs,antiviral chemotherapeutics, and cytostatics, i.e., antineoplasticagents or cytotoxic drugs, such as alkylating agents, e.g.,mechlorethamine hydrochloride (nitrogen mustard, mustargen, HN2),cyclophosphamide (Cytovan, Endoxana), ifosfamide (IFEX), chlorambucil(Leukeran), Melphalan (phenylalanine mustard, L-sarcolysin, Alkeran,L-PAM), busulfan (Myleran), Thiotepa (triethylenethiophosphoramide),carmustine (BiCNU, BCNU), lomustine (CeeNU, CCNU), streptozocin(Zanosar); plant alkaloids, e.g., vincristine (Oncovin), vinblastine(Velban, Velbe), paclitaxel (Taxol); antimetabolites, e.g., methotrexate(MTX), mercaptopurine (Purinethol, 6-MP), thioguanine (6-TG),fluorouracil (5-FU), cytarabine (Cytosar-U, Ara-C), azacitidine(Mylosar, 5-AZA); antibiotics, e.g., dactinomycin (Actinomycin D,Cosmegen), doxorubicin (Adriamycin), daunorubicin (duanomycin,Cerubidine), idarubicin (Idamycin), bleomycin (Blenoxane), picamycin(Mithramycin, Mithracin), mitomycin (Mutamycin), and other anticellularproliferative agents, e.g., hydroxyurea (Hydrea), procarbazine(Mutalane), dacarbazine (DTIC-Dome), cisplatin (Platinol) carboplatin(Paraplatin), asparaginase (Elspar), etoposide (VePesid, VP-16-213),amsarcrine (AMSA, m-AMSA), mitotane (Lysodren), or mitoxantrone(Novatrone).

In various embodiments, the effector moiety is derived from one or more:anti-inflammatory agents; antibiotics, such as: aminoglycosides, e.g.,amikacin, apramycin, arbekacin, bambermycins, butirosin, dibekacin,dihydrostreptomycin, fortimicin, gentamicin, isepamicin, kanamycin,micronomcin, neomycin, netilmicin, paromycin, ribostamycin, sisomicin,spectinomycin, streptomycin, tobramycin, trospectomycin; amphenicols,e.g., azidamfenicol, chloramphenicol, florfenicol, and theimaphenicol;ansamycins, e.g., rifamide, rifampin, rifamycin, rifapentine, rifaximin;β-lactams, e.g., carbacephems, carbapenems, cephalosporins, cehpamycins,monobactams, oxaphems, penicillins; lincosamides, e.g., clinamycin,lincomycin; macrolides, e.g., clarithromycin, dirthromycin,erythromycin; polypeptides, e.g., amphomycin, bacitracin, capreomycin;tetracyclines, e.g., apicycline, chlortetracycline, clomocycline;synthetic antibacterial agents, such as 2,4-diaminopyrimidines,nitrofurans, quinolones and analogs thereof, sulfonamides, or sulfones.

In various embodiments, the effector moiety is derived from one or more:antifungal agents, such as: polyenes, e.g., amphotericin B, candicidin,dermostatin, filipin, fungichromin, hachimycin, hamycin, lucensomycin,mepartricin, natamycin, nystatin, pecilocin, perimycin; syntheticantifungals, such as allylamines, e.g., butenafine, naftifine,terbinafine; imidazoles, e.g., bifonazole, butoconazole, chlordantoin,chlormidazole, thiocarbamates, e.g., tolciclate, triazoles, e.g.,fluconazole, itraconazole, or terconazole.

In various embodiments, the effector moiety is derived from one or more:anthelmintics, such as: arecoline, aspidin, aspidinol, dichlorophene,embelin, kosin, napthalene, niclosamide, pelletierine, quinacrine,alantolactone, amocarzine, amoscanate, ascaridole, bephenium,bitoscanate, carbon tetrachloride, carvacrol, cyclobendazole, ordiethylcarbamazine.

In various embodiments, the effector moiety is derived from one or more:antimalarials, such as: acedapsone, amodiaquin, arteether, artemether,artemisinin, artesunate, atovaquone, bebeerine, berberine, chirata,chlorguanide, chloroquine, chlorprogaunil, cinchona, cinchonidine,cinchonine, cycloguanil, gentiopicrin, halofantrine, hydroxychloroquine,mefloquine hydrochloride, 3-methylarsacetin, pamaquine, plasmocid,primaquine, pyrimethamine, quinacrine, quinidine, quinine, quinocide,quinoline, or dibasic sodium arsenate.

In various embodiments, the effector moiety is derived from one or more:antiprotozoan agents, such as: acranil, tinidazole, ipronidazole,ethylstibamine, pentamidine, acetarsone, aminitrozole, anisomycin,nifuratel, tinidazole, benzidazole, or suramin.

In various embodiments, the effector moiety includes one or more of:docetaxel or paclitaxel; BEZ235; temsirolimus; PLX4032; cisplatin;AZD8055; and crizotinib.

In various embodiments, the effector moiety includes a topotecan oririnotecan.

In various embodiments, the cytotoxic moiety is not suitable foradministration alone. The cytotoxic moiety can be unsuitable foradministration alone due to toxicity. The cytotoxic moiety can beunsuitable for administration alone due to undesired targeting or a lackof targeting.

In various embodiments, the binding moiety and the effector moiety arecovalently attached. The binding moiety and the effector moiety can becovalently attached, for example by a linker. The linker can comprise acleavable linker. The cleavable linker can comprise an enzymaticallycleavable linker. The linker can be selected from the group consistingof disulfide, carbamate, amide, ester, and ether linkers.

In various embodiments, the SDC-TRAP has a molecular weight of less thanabout 1600 Dalton. For example, the SDC-TRAP molecular weight can beless than about 1600, 1550, 1500, 1450, 1400, 1350, 1300, 1250, 1200,1150, 1100, 1050, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550,500, 450, 400, 350, 300, 250, or 200 Dalton.

In various embodiments, the binding moiety has a molecular weight ofless than about 800 Dalton. For example, the binding moiety molecularweight can be less than about 800, 750, 700, 650, 600, 550, 500, 450,400, 350, 300, 250, 200, 150, or 100 Dalton.

In various embodiments, the effector moiety has a molecular weight ofless than about 800 Dalton. For example, the effector moiety molecularweight can be less than about 800, 750, 700, 650, 600, 550, 500, 450,400, 350, 300, 250, 200, 150, or 100 Dalton.

In various embodiments, the binding moiety and the effector moiety areapproximately equal in size. For example, the binding moiety and theeffector moiety can have less than about a 25, 50, 75, 100, 125, 150,175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 Dalton difference inmolecular weight.

In various embodiments, the binding moiety has a high affinity for amolecular target. For example, the binding moiety has a high affinityfor a molecular target that is a K_(d) of 50, 100, 150, 200, 250, 300,350, 400 nM or higher.

In various embodiments, when administered to a subject, the SDC-TRAP ispresent at a ratio of about 2:1, 5:1, 10:1, 25:1, 50:1, 75:1, 100:1,150:1, 200:1, 250:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1,1000:1, or greater. The ratio can be, for example, at 1, 2, 3, 4, 5, 6,7, 8, 12, 24, 48, 72, or more hours from administration.

In various embodiments, the SDC-TRAP is present in target cells and/ortissue for at least 24 hours. The SDC-TRAP can be present in cancercells for longer, for example, for at least 48, 72, 96, or 120 hours.

In various embodiments, the effector moiety is released for a period ofat least 6 hours. The effector moiety can be released for a longerperiod, for example, for at least 12, 24, 48, 72, 96, or 120 hours.

In various embodiments, the effector moiety is selectively releasedinside a target cell and/or tissue.

In various embodiments, the present invention provides SDC-TRAPmolecules comprising a binding moiety is an inhibitor of a targetprotein but that is ineffective as a therapeutic agent when administeredalone. In these, and in other embodiments, the SDC-TRAP may facilitatean additive or synergistic effect between the binding moiety andeffector moiety.

In various embodiments, the present invention provides SDC-TRAPmolecules SDC-TRAP molecules selected from the group consisting of:SDC-TRAP-0008, SDC-TRAP-0015, SDC-TRAP-0016, SDC-TRAP-0017,SDC-TRAP-0018, SDC-TRAP-0019, SDC-TRAP-0020, SDC-TRAP-0021,SDC-TRAP-0022, SDC-TRAP-0010, SDC-TRAP-0023, SDC-TRAP-0027,SDC-TRAP-0028, SDC-TRAP-0029, SDC-TRAP-0031, SDC-TRAP-0024,SDC-TRAP-0025, SDC-TRAP-0033, SDC-TRAP-0037, SDC-TRAP-0038,SDC-TRAP-0039, SDC-TRAP-0040, SDC-TRAP-0041, SDC-TRAP-0042,SDC-TRAP-0043, SDC-TRAP-0044, SDC-TRAP-0045, SDC-TRAP-0046,SDC-TRAP-0047, SDC-TRAP-0048, SDC-TRAP-0049, SDC-TRAP-0050,SDC-TRAP-0051, SDC-TRAP-0063, SDC-TRAP-0178, SDC-TRAP-0069,SDC-TRAP-0211, SDC-TRAP-0098, SDC-TRAP-0198, SDC-TRAP-0199,SDC-TRAP-0219, SDC-TRAP-0200, SDC-TRAP-0068, SDC-TRAP-0093,SDC-TRAP-0117, SDC-TRAP-0201, SDC-TRAP-0204, SDC-TRAP-0171,SDC-TRAP-0196, SDC-TRAP-0003, SDC-TRAP-0004, SDC-TRAP-0005,SDC-TRAP-0006, SDC-TRAP-0030, SDC-TRAP-0032, SDC-TRAP-0034,SDC-TRAP-0035, SDC-TRAP-0036, SDC-TRAP-0224, SDC-TRAP-0225,SDC-TRAP-0226, SDC-TRAP-0227, SDC-TRAP-0228, SDC-TRAP-0223,SDC-TRAP-0002, SDC-TRAP-0056, SDC-TRAP-0052, SDC-TRAP-0064,SDC-TRAP-0172, SDC-TRAP-0180, SDC-TRAP-0184, SDC-TRAP-0185,SDC-TRAP-0186, SDC-TRAP-0118, SDC-TRAP-0009, SDC-TRAP-0013,SDC-TRAP-0137, SDC-TRAP-0150, SDC-TRAP-0151, SDC-TRAP-0153,SDC-TRAP-0134, SDC-TRAP-0139, SDC-TRAP-0138, SDC-TRAP-0142,SDC-TRAP-0105, SDC-TRAP-0108, SDC-TRAP-0126, SDC-TRAP-0132,SDC-TRAP-0127, SDC-TRAP-0133, SDC-TRAP-0135, SDC-TRAP-0140,SDC-TRAP-0136, SDC-TRAP-0231, SDC-TRAP-0147, SDC-TRAP-0165,SDC-TRAP-0163, SDC-TRAP-0164, SDC-TRAP-0166, SDC-TRAP-0188,SDC-TRAP-0189, SDC-TRAP-0190, SDC-TRAP-0191, SDC-TRAP-0192,SDC-TRAP-0193, SDC-TRAP-0122, SDC-TRAP-0123, SDC-TRAP-0124,SDC-TRAP-0125, SDC-TRAP-0155, SDC-TRAP-0156, SDC-TRAP-0157,SDC-TRAP-0160, SDC-TRAP-0167, SDC-TRAP-0168, SDC-TRAP-0170,SDC-TRAP-0171, SDC-TRAP-0182, SDC-TRAP-0187, SDC-TRAP-0109,SDC-TRAP-0110, SDC-TRAP-0114, SDC-TRAP-0115, SDC-TRAP-0116,SDC-TRAP-0119, SDC-TRAP-0120, SDC-TRAP-0121, SDC-TRAP-0128,SDC-TRAP-0129, SDC-TRAP-0131, SDC-TRAP-0149, SDC-TRAP-0152,SDC-TRAP-0168, SDC-TRAP-0173, SDC-TRAP-0174, SDC-TRAP-0175,SDC-TRAP-0176, SDC-TRAP-0177, SDC-TRAP-0178, SDC-TRAP-0194,SDC-TRAP-0195, SDC-TRAP-0078, SDC-TRAP-0082, SDC-TRAP-0093,SDC-TRAP-0102, SDC-TRAP-0103, SDC-TRAP-0130, SDC-TRAP-0011,SDC-TRAP-0012, SDC-TRAP-0014, SDC-TRAP-0065, SDC-TRAP-0066,SDC-TRAP-0084, SDC-TRAP-0086, SDC-TRAP-0088, SDC-TRAP-0087,SDC-TRAP-0089, SDC-TRAP-0090, SDC-TRAP-0091, SDC-TRAP-0092,SDC-TRAP-0104, SDC-TRAP-0106, SDC-TRAP-0107, SDC-TRAP-0145,SDC-TRAP-0207, SDC-TRAP-0206, SDC-TRAP-0205, SDC-TRAP-0208,SDC-TRAP-0209, SDC-TRAP-0210, SDC-TRAP-0213, SDC-TRAP-0214,SDC-TRAP-0215, SDC-TRAP-0216, SDC-TRAP-0217, SDC-TRAP-0218,SDC-TRAP-0067, SDC-TRAP-0070, SDC-TRAP-0077, SDC-TRAP-0079,SDC-TRAP-0081, SDC-TRAP-0083, SDC-TRAP-0094, SDC-TRAP-0095,SDC-TRAP-0101, SDC-TRAP-0220, SDC-TRAP-0026, SDC-TRAP-0055,SDC-TRAP-0057, SDC-TRAP-0058, SDC-TRAP-0060, SDC-TRAP-0061,SDC-TRAP-0071, SDC-TRAP-0072, SDC-TRAP-0073, SDC-TRAP-0074,SDC-TRAP-0075, SDC-TRAP-0076, SDC-TRAP-0097, SDC-TRAP-0100,SDC-TRAP-0111, SDC-TRAP-0112, SDC-TRAP-0113, SDC-TRAP-0154,SDC-TRAP-0169, SDC-TRAP-0181, SDC-TRAP-0202, SDC-TRAP-0203,SDC-TRAP-0221, SDC-TRAP-0222, SDC-TRAP-0148, SDC-TRAP-0159,SDC-TRAP-0099, SDC-TRAP-0158, SDC-TRAP-0085, SDC-TRAP-0232,SDC-TRAP-0233 and SDC-TRAP-0234.

In various embodiments, the present invention features a method fortreating a subject having a disease or disorder comprising administeringa therapeutically effective amount of at least one SDC-TRAP to thesubject, thereby treating the disease or disorder, wherein the SDC-TRAPcomprises an SDC-TRAP described herein.

In one embodiment, the disease or disorder is selected from the groupconsisting of: cancer, actinic keratosis, chronic bronchitis and asthma.

In various embodiments the present invention features a method fortreating a subject having a cancer comprising administering atherapeutically effective amount of at least one SDC-TRAP to thesubject, thereby treating the cancer, wherein the SDC-TRAP comprises anSDC-TRAP described herein.

The present invention is described in further detail by the figures andexamples below, which are used only for illustration purposes and arenot limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows how an illustrative Hsp90-targeting moiety may be suitablymodified at one or more positions to enhance the physical,pharmacokinetic, or pharmacodynamic properties of the conjugate.

FIG. 2 illustrates an embodiment of a pharmaceutical conjugate havingtwo effector moieties.

FIG. 3 illustrates an example where the mean concentration of ganetespibin plasma is about 10 times higher than that in RBC at 5 min time point.

FIG. 4 shows the change in tumor volume following treatment withSDC-TRAP-0063, compared to effector moiety irinotecan and vehiclecontrol in an HCT-116 colon cancer model.

FIG. 5 shows the change in animal body weight following treatment withSDC-TRAP-0063, compared to effector moiety irinotecan and vehiclecontrol in an HCT-116 colon cancer model.

FIG. 6 shows the change in tumor volume following treatment withSDC-TRAP-0063, compared to effector moiety irinotecan and vehiclecontrol in an MCF-7 breast cancer model.

FIG. 7 shows the change in animal body weight following treatment withSDC-TRAP-0063, compared to effector moiety irinotecan and vehiclecontrol in an MCF-7 breast cancer model.

FIG. 8 demonstrates a dose-dependent decrease in tumor volume comparedto binding moiety or effector moiety alone.

FIGS. 9, 10, and 11 show that following SDC-TRAP intravenous injection,binding moiety and effector moiety accumulate and persist in tumor, butrapidly diminish in plasma and heart in three mouse strains.

FIG. 12 illustrates the stability of seven SDC-TRAP species in mouseplasma.

FIG. 13 illustrates the stability of five additional SDC-TRAP speciesplus effector moiety SN-38 in mouse plasma and cell culture media.

FIG. 14 depicts the stability of SDC-TRAP-0063 and SN-38 alone.

FIGS. 15 A-C depict the tissue distribution of SDC-TRAP-0063, and itsdegradation products DP-1 and SN-38, respectively in plasma, tumor andheart.

Other features and advantages of the instant invention will be apparentfrom the following detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides molecules including an effector moietyconjugated to a binding moiety that directs the effector moiety to abiological target of interest. The molecules of the invention allow forselective targeting of an effector moiety by trapping the molecules ofthe invention in a desired cell, e.g., a cancer cell. The molecules canbe described as Small molecule Drug Congugates that are TRAPpedintracellularly (SDC-TRAP), due to their selective binding to highconcentration intracellular proteins. In order for the molecules of theinvention to be trapped within the cells of interest, the bindingmoieties that are part of the SDC-TRAP molecules interact with proteinsthat are overexpressed in targeted cells. In exemplary embodiments, theproteins that are overexpressed are characteristic of a particulardisease or disorder. Accordingly, the present invention providescompositions, kits, and methods (e.g., therapeutic, diagnostic, andimaging) that include the molecules of the invention.

In one embodiment of the invention, SDC-TRAPs allow for the delivery ofa effector molecule that would otherwise be unsuitable foradministration alone due to toxicity and/or undesired systemic effects.Using the targeted delivery molecules described herein (SDC-TRAPs)allows for effector moieties that are too toxic to administer by currentmethods to be dosed at lower levels thereby allowing the toxic effectorto be targeted to specific diseased cells at sub-toxic levels.

In various exemplary aspects and embodiments, the present inventionprovides compounds for treating cancer. For example, an SDC-TRAP cancomprise an Hsp90 binding moiety (i.e., targeting Hsp90, which isoverexpressed in cancer cells compared to normal cells) and an effectormoiety (e.g., the Hsp90 binding moiety can be an Hsp90 inhibitor that isconjugated to a cytotoxic agent). As indicated above, the invention isexemplified herein in terms of Hsp90-targeted binding moieties andcytotoxic agents. Other binding moieties that are contemplated,mentioned or described herein are intended to be included within thescope of the invention.

In various aspects and embodiments, the present invention provides anSDC-TRAP comprising a binding moiety and an effector moiety, wherein theSDC-TRAP molecule is able to enter a cell by passive transport. Theability of an SDC-TRAP to enter a cell by passive transport can be aresult of one or more unique chemical properties of the SDC-TRAP (e.g.,size, weight, charge, polarity, hydrophobicity, etc.) and can facilitatethe delivery and/or action of the SDC-TRAP. The ability of an SDC-TRAPto enter a cell by passive transport is a functional property, whichalong with its physico-chemical properties, differentiates SDC-TRAPsfrom other targeted molecules such as antibody-drug conjugates.

In various aspects and embodiments, the present invention provides anSDC-TRAP comprising a binding moiety and an effector moiety, whereinSDC-TRAP molecule is able to enter a cell by active transport. Theability of an SDC-TRAP to enter a cell by active transport can be aresult of one or more unique chemical properties of the SDC-TRAP and canfacilitate the delivery and/or action of the SDC-TRAP. Example ofSDC-TRAP active transport can include, for example, endocytosis,phagocytosis, pinocytosis, and exocytosis.

In various aspects and embodiments, the present invention provides anSDC-TRAP having a molecular weight of less than about 1600 Dalton (e.g.,less than about 1600, 1550, 1500, 1450, 1400, 1350, 1300, 1250, 1200,1150, 1100, 1050, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550,500, 450, 400, 350, 300, 250, 200, etc.). Similarly, in various aspectsand embodiments, the present invention provides a binding moiety havinga molecular weight of less than about 800 Dalton (e.g., less than about800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150,100, etc.) and/or an effector moiety having a molecular weight of lessthan about 800 Dalton (e.g., less than about 800, 750, 700, 650, 600,550, 500, 450, 400, 350, 300, 250, 200, 150, 100, etc.). The overallmolecular weight of an SDC-TRAP, and the individual weights of a bindingmoiety, effector moiety, and any linking moiety, can affect transport ofthe SDC-TRAP. In various examples, it has been observed that lowermolecular weights can facilitate delivery and/or activity of anSDC-TRAP.

In various aspects and embodiments, the present invention provides anSDC-TRAP comprising an Hsp90 binding moiety and an effector moiety,wherein the Hsp90 binding moiety and the effector moiety areapproximately equal in size (e.g., the Hsp90 binding moiety and theeffector moiety have less than about a 25, 50, 75, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 375, 400, etc. Dalton difference inmolecular weight.) In various examples, it has been observed that lowerdifferences in molecular weight can facilitate delivery and/or activityof an SDC-TRAP.

In various aspects and embodiments, the present invention provides anSDC-TRAP comprising a target protein-interacting binding moiety. Atarget protein-interacting binding moiety can selectively interact withany one or more domains of a target protein. For example, where a targetprotein is Hsp90, the binding moiety can be an Hsp90 binding moiety thatinteracts with the N-terminal domain of Hsp90, the C-terminal domain ofHsp90, and/or the middle domain of Hsp90. Selective interaction with anyone or more domains of a target protein can advantageously increasespecificity and/or increase the concentration of molecular targetswithin a target tissue and/or cell.

In various aspects and embodiments, the present invention provides anSDC-TRAP comprising a binding moiety having a high affinity for amolecular target (e.g., a K_(d) of 50, 100, 150, 200, 250, 300, 350, 400nM or higher). For example, where a binding moiety is an Hsp90 bindingmoiety, the Hsp90 binding moiety can have a K_(d) of 50, 100, 150, 200,250, 300, 350, 400 nM or higher. A binding moiety having a high affinityfor a molecular target can advantageously improve targeting and/orincrease the resonance time of the SDC-TRAP in a target cell and/ortissue.

In various aspects and embodiments, the present invention provides anSDC-TRAP comprising a binding moiety (e.g., Hsp90 binding moiety) and aneffector moiety, wherein when administered to a subject the SDC-TRAP ispresent at a ratio of about 2:1 in tumor cells compared to plasma. Theratio can be higher, for example, about 5:1, 10:1, 25:1, 50:1, 75:1,100:1, 150:1, 200:1, 250:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1,900:1, 1000:1, or greater. In various aspects and embodiments, the ratiois at 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, 48, 72, or more hours fromadministration. The effectiveness of targeting can be reflected in theratio of SDC-TRAP in a target cell and/or tissue compared to plasma.

In various aspects and embodiments, the present invention provides anSDC-TRAP comprising a binding moiety (e.g., Hsp90 binding moiety) and aneffector moiety, wherein the SDC-TRAP is present in target (e.g.,cancer) cells for at least 24 hours. The SDC-TRAP can be present incancer cells for longer, for example, for at least 48, 72, 96, or 120hours. It can be advantageous for an SDC-TRAP to be present in targetcells for longer periods of time to increase the therapeutic effect of agiven dose of SDC-TRAP and/or increase an interval betweenadministrations of SDC-TRAP.

In various aspects and embodiments, the present invention provides anSDC-TRAP comprising a binding moiety (e.g., Hsp90 binding moiety) and aneffector moiety, wherein the effector moiety is released for a period ofat least 6 hours. The effector moiety can be released for a longerperiod, for example, for at least 12, 24, 48, 72, 96, or 120 hours.Selective release can be used to control, delay, and/or extend theperiod of release of an effector moiety and, therefore, increase thetherapeutic effect of a given dose of SDC-TRAP, decrease the undesiredside effects of a given dose of SDC-TRAP, and/or increase an intervalbetween administrations of SDC-TRAP.

In various aspects and embodiments, the present invention provides anSDC-TRAP comprising an Hsp90 binding moiety and an effector moiety,wherein the effector moiety is selectively released inside a target(e.g., cancer) cell. Selective release can be achieved, for example, bya cleavable linker (e.g., an enzymatically cleavable linker). Selectiverelease can be used to decrease undesired toxicity and/or unwanted sideeffects. For example, an SDC-TRAP can be designed where an effectormoiety such is inactive (or relatively inactive) in a conjugated form,but active (or more active) after it is selectively released inside atarget (e.g., cancer) cell.

In various aspects and embodiments, the present invention provides anSDC-TRAP comprising a binding moiety (e.g., Hsp90 binding moiety) and aneffector moiety, wherein the SDC-TRAP allows for the use of an effectormoiety that is otherwise toxic or unfit for administration to a subject.The effector moiety can be unfit for administration to a subject becauseof undesired toxicity. In such cases, a strategy such as selectiverelease may be used to address the undesired toxicity. The effectormoiety can be unfit for administration to a subject because of undesiredtargeting or a lack of targeting. Targeting can address such problems,for example, by minimizing systemic toxicity while maximizing localtoxicity at a target (e.g., a tumor).

In various aspects and embodiments, the present invention provides anSDC-TRAP comprising a binding moiety (e.g., Hsp90 binding moiety) and aneffector moiety, wherein the binding moiety is an inhibitor (e.g., Hsp90inhibitor) that is ineffective as a therapeutic agent when administeredalone. In such cases, the SDC-TRAP may facilitate an additive orsynergistic effect between the binding moiety and effector moiety,thereby advantageously improving the efficacy and/or reducing the sideeffects of a therapy.

In order that the present invention may be more readily understood,certain terms are first defined. In addition, it should be noted thatwhenever a value or range of values of a parameter are recited, it isintended that values and ranges intermediate to the recited values arealso intended to be part of this invention. Unless defined otherwise,all technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting.

Definitions

The articles “a,” “an,” and “the” are used herein to refer to one or tomore than one (i.e. to at least one) of the grammatical object of thearticle unless otherwise clearly indicated by contrast. By way ofexample, “an element” means one element or more than one element.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to.”

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

The term “such as” is used herein to mean, and is used interchangeably,with the phrase “such as but not limited to.”

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein can be modified by theterm about.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

The recitation of a listing of chemical group(s) in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

As used herein, the term “subject” refers to human and non-humananimals, including veterinary subjects. The term “non-human animal”includes all vertebrates, e.g., mammals and non-mammals, such asnon-human primates, mice, rabbits, sheep, dog, cat, horse, cow,chickens, amphibians, and reptiles. In a preferred embodiment, thesubject is a human and may be referred to as a patient.

As used herein, the terms “treat,” “treating” or “treatment” refer,preferably, to an action to obtain a beneficial or desired clinicalresult including, but not limited to, alleviation or amelioration of oneor more signs or symptoms of a disease or condition, diminishing theextent of disease, stability (i.e., not worsening) state of disease,amelioration or palliation of the disease state, diminishing rate of ortime to progression, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival in the absence of treatment.Treatment does not need to be curative.

A “therapeutically effective amount” is that amount sufficient to treata disease in a subject. A therapeutically effective amount can beadministered in one or more administrations.

By “diagnosing” and the like, as used herein, refers to a clinical orother assessment of the condition of a subject based on observation,testing, or circumstances for identifying a subject having a disease,disorder, or condition based on the presence of at least one indicator,such as a sign or symptom of the disease, disorder, or condition.Typically, diagnosing using the method of the invention includes theobservation of the subject for multiple indicators of the disease,disorder, or condition in conjunction with the methods provided herein.Diagnostic methods provide an indicator that a disease is or is notpresent. A single diagnostic test typically does not provide adefinitive conclusion regarding the disease state of the subject beingtested.

The terms “administer,” “administering” or “administration” include anymethod of delivery of a pharmaceutical composition or agent into asubject's system or to a particular region in or on a subject. Incertain embodiments of the invention, an agent is administeredintravenously, intramuscularly, subcutaneously, intradermally,intranasally, orally, transcutaneously, or mucosally. In a preferredembodiment, an agent is administered intravenously. Administering anagent can be performed by a number of people working in concert.Administering an agent includes, for example, prescribing an agent to beadministered to a subject and/or providing instructions, directly orthrough another, to take a specific agent, either by self-delivery,e.g., as by oral delivery, subcutaneous delivery, intravenous deliverythrough a central line, etc.; or for delivery by a trained professional,e.g., intravenous delivery, intramuscular delivery, intratumoraldelivery, etc.

As used herein, the term “survival” refers to the continuation of lifeof a subject which has been treated for a disease or condition, e.g.,cancer. The time of survival can be defined from an arbitrary point suchas time of entry into a clinical trial, time from completion or failureor an earlier treatment regimen, time from diagnosis, etc.

As used herein, the term “recur” refers to the re-growth of tumor orcancerous cells in a subject in whom primary treatment for the tumor hasbeen administered. The tumor may recur in the original site or inanother part of the body. In one embodiment, a tumor that recurs is ofthe same type as the original tumor for which the subject was treated.For example, if a subject had an ovarian cancer tumor, was treated andsubsequently developed another ovarian cancer tumor, the tumor hasrecurred. In addition, a cancer can recur in or metastasize to adifferent organ or tissue than the one where it originally occurred.

As used herein, the terms “identify” or “select” refer to a choice inpreference to another. In other words, to identify a subject or select asubject is to perform the active step of picking out that particularsubject from a group and confirming the identity of the subject by nameor other distinguishing feature.

As used herein, the term “benefit” refers to something that isadvantageous or good, or an advantage. Similarly, the term “benefiting,”as used herein, refers to something that improves or advantages. Forexample, a subject will benefit from treatment if they exhibit adecrease in at least one sign or symptom of a disease or condition(e.g., tumor shrinkage, decrease in tumor burden, inhibition or decreaseof metastasis, improving quality of life (“QOL”), if there is a delay oftime to progression (“TTP”), if there is an increase of overall survival(“OS”), etc.), or if there is a slowing or stopping of diseaseprogression (e.g., halting tumor growth or metastasis, or slowing therate of tumor growth or metastasis). A benefit can also include animprovement in quality of life, or an increase in survival time orprogression free survival.

The terms “cancer” or “tumor” are well known in the art and refer to thepresence, e.g., in a subject, of cells possessing characteristicstypical of cancer-causing cells, such as uncontrolled proliferation,immortality, metastatic potential, rapid growth and proliferation rate,decreased cell death/apoptosis, and certain characteristic morphologicalfeatures. Cancer cells are often in the form of a solid tumor. However,cancer also includes non-solid tumors, e.g., blood tumors, e.g.,leukemia, wherein the cancer cells are derived from bone marrow. As usedherein, the term “cancer” includes pre-malignant as well as malignantcancers. Cancers include, but are not limited to, acoustic neuroma,acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia(monocytic, myeloblastic, adenocarcinoma, angiosarcoma, astrocytoma,myelomonocytic and promyelocytic), acute T-cell leukemia, basal cellcarcinoma, bile duct carcinoma, bladder cancer, brain cancer, breastcancer, bronchogenic carcinoma, cervical cancer, chondrosarcoma,chordoma, choriocarcinoma, chronic leukemia, chronic lymphocyticleukemia, chronic myelocytic (granulocytic) leukemia, chronicmyelogenous leukemia, colon cancer, colorectal cancer,craniopharyngioma, cystadenocarcinoma, diffuse large B-cell lymphoma,Burkitt's lymphoma, dysproliferative changes (dysplasias andmetaplasias), embryonal carcinoma, endometrial cancer,endotheliosarcoma, ependymoma, epithelial carcinoma, erythroleukemia,esophageal cancer, estrogen-receptor positive breast cancer, essentialthrombocythemia, Ewing's tumor, fibrosarcoma, follicular lymphoma, germcell testicular cancer, glioma, heavy chain disease, hemangioblastoma,hepatoma, hepatocellular cancer, hormone insensitive prostate cancer,leiomyosarcoma, liposarcoma, lung cancer, lymphagioendotheliosarcoma,lymphangiosarcoma, lymphoblastic leukemia, lymphoma (Hodgkin's andnon-Hodgkin's), malignancies and hyperproliferative disorders of thebladder, breast, colon, lung, ovaries, pancreas, prostate, skin, anduterus, lymphoid malignancies of T-cell or B-cell origin, leukemia,lymphoma, medullary carcinoma, medulloblastoma, melanoma, meningioma,mesothelioma, multiple myeloma, myelogenous leukemia, myeloma,myxosarcoma, neuroblastoma, non-small cell lung cancer,oligodendroglioma, oral cancer, osteogenic sarcoma, ovarian cancer,pancreatic cancer, papillary adenocarcinomas, papillary carcinoma,pinealoma, polycythemia vera, prostate cancer, rectal cancer, renal cellcarcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, sebaceous glandcarcinoma, seminoma, skin cancer, small cell lung carcinoma, solidtumors (carcinomas and sarcomas), small cell lung cancer, stomachcancer, squamous cell carcinoma, synovioma, sweat gland carcinoma,thyroid cancer, Waldenstrom's macroglobulinemia, testicular tumors,uterine cancer, and Wilms' tumor. Other cancers include primary cancer,metastatic cancer, oropharyngeal cancer, hypopharyngeal cancer, livercancer, gall bladder cancer, bile duct cancer, small intestine cancer,urinary tract cancer, kidney cancer, urothelium cancer, female genitaltract cancer, uterine cancer, gestational trophoblastic disease, malegenital tract cancer, seminal vesicle cancer, testicular cancer, germcell tumors, endocrine gland tumors, thyroid cancer, adrenal cancer,pituitary gland cancer, hemangioma, sarcoma arising from bone and softtissues, Kaposi's sarcoma, nerve cancer, ocular cancer, meningialcancer, glioblastomas, neuromas, neuroblastomas, Schwannomas, solidtumors arising from hematopoietic malignancies such as leukemias,metastatic melanoma, recurrent or persistent ovarian epithelial cancer,fallopian tube cancer, primary peritoneal cancer, gastrointestinalstromal tumors, colorectal cancer, gastric cancer, melanoma,glioblastoma multiforme, non-squamous non-small-cell lung cancer,malignant glioma, epithelial ovarian cancer, primary peritoneal serouscancer, metastatic liver cancer, neuroendocrine carcinoma, refractorymalignancy, triple negative breast cancer, HER2-amplified breast cancer,nasopharageal cancer, oral cancer, biliary tract, hepatocellularcarcinoma, squamous cell carcinomas of the head and neck (SCCHN),non-medullary thyroid carcinoma, recurrent glioblastoma multiforme,neurofibromatosis type 1, CNS cancer, liposarcoma, leiomyosarcoma,salivary gland cancer, mucosal melanoma, acral/lentiginous melanoma,paraganglioma, pheochromocytoma, advanced metastatic cancer, solidtumor, triple negative breast cancer, colorectal cancer, sarcoma,melanoma, renal carcinoma, endometrial cancer, thyroid cancer,rhabdomysarcoma, multiple myeloma, ovarian cancer, glioblastoma,gastrointestinal stromal tumor, mantle cell lymphoma, and refractorymalignancy.

“Solid tumor,” as used herein, is understood as any pathogenic tumorthat can be palpated or detected using imaging methods as an abnormalgrowth having three dimensions. A solid tumor is differentiated from ablood tumor such as leukemia. However, cells of a blood tumor arederived from bone marrow; therefore, the tissue producing the cancercells is a solid tissue that can be hypoxic.

“Tumor tissue” is understood as cells, extracellular matrix, and othernaturally occurring components associated with the solid tumor.

As used herein, the term “isolated” refers to a preparation that issubstantially free (e.g., 50%, 60%, 70%, 80%, 90% or more, by weight)from other proteins, nucleic acids, or compounds associated with thetissue from which the preparation is obtained.

The term “sample” as used herein refers to a collection of similarfluids, cells, or tissues isolated from a subject. The term “sample”includes any body fluid (e.g., urine, serum, blood fluids, lymph,gynecological fluids, cystic fluid, ascetic fluid, ocular fluids, andfluids collected by bronchial lavage and/or peritoneal rinsing),ascites, tissue samples (e.g., tumor samples) or a cell from a subject.Other subject samples include tear drops, serum, cerebrospinal fluid,feces, sputum, and cell extracts. In one embodiment, the sample isremoved from the subject. In a particular embodiment, the sample isurine or serum. In another embodiment, the sample does not includeascites or is not an ascites sample. In another embodiment, the sampledoes not include peritoneal fluid or is not peritoneal fluid. In oneembodiment, the sample comprises cells. In another embodiment, thesample does not comprise cells. Samples are typically removed from thesubject prior to analysis. However, tumor samples can be analyzed in thesubject, for example, using imaging or other detection methods.

The term “control sample,” as used herein, refers to any clinicallyrelevant comparative sample, including, for example, a sample from ahealthy subject not afflicted with cancer, a sample from a subjecthaving a less severe or slower progressing cancer than the subject to beassessed, a sample from a subject having some other type of cancer ordisease, a sample from a subject prior to treatment, a sample ofnon-diseased tissue (e.g., non-tumor tissue), a sample from the sameorigin and close to the tumor site, and the like. A control sample canbe a purified sample, protein, and/or nucleic acid provided with a kit.Such control samples can be diluted, for example, in a dilution seriesto allow for quantitative measurement of analytes in test samples. Acontrol sample may include a sample derived from one or more subjects. Acontrol sample may also be a sample made at an earlier time point fromthe subject to be assessed. For example, the control sample could be asample taken from the subject to be assessed before the onset of thecancer, at an earlier stage of disease, or before the administration oftreatment or of a portion of treatment. The control sample may also be asample from an animal model, or from a tissue or cell lines derived fromthe animal model, of the cancer. The level in a control sample thatconsists of a group of measurements may be determined, e.g., based onany appropriate statistical measure, such as, for example, measures ofcentral tendency including average, median, or modal values.

As used herein, the term “obtaining” is understood herein asmanufacturing, purchasing, or otherwise coming into possession of.

As used herein, the term “identical” or “identity” is used herein inrelation to amino acid or nucleic acid sequences refers to any gene orprotein sequence that bears at least 30% identity, more preferably 40%,50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, and most preferably 95%, 96%, 97%, 98%, 99% ormore identity to a known gene or protein sequence over the length of thecomparison sequence. Protein or nucleic acid sequences with high levelsof identity throughout the sequence can be said to be homologous. A“homologous” protein can also have at least one biological activity ofthe comparison protein. In general, for proteins, the length ofcomparison sequences will be at least 10 amino acids, preferably 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 150, 175, 200, 250, or at least 300amino acids or more. For nucleic acids, the length of comparisonsequences will generally be at least 25, 50, 100, 125, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 800, or at least 850nucleotides or more.

As used herein, “detecting,” “detection” and the like are understoodthat an assay performed for identification of a specific analyte in asample. The amount of analyte or activity detected in the sample can benone or below the level of detection of the assay or method.

The terms “modulate” or “modulation” refer to upregulation (i.e.,activation or stimulation), downregulation (i.e., inhibition orsuppression) of a level, or the two in combination or apart. A“modulator” is a compound or molecule that modulates, and may be, e.g.,an agonist, antagonist, activator, stimulator, suppressor, or inhibitor.

The term “expression” is used herein to mean the process by which apolypeptide is produced from DNA. The process involves the transcriptionof the gene into mRNA and the translation of this mRNA into apolypeptide. Depending on the context in which used, “expression” mayrefer to the production of RNA, or protein, or both.

The terms “level of expression of a gene” or “gene expression level”refer to the level of mRNA, as well as pre-mRNA nascent transcript(s),transcript processing intermediates, mature mRNA(s) and degradationproducts, or the level of protein, encoded by the gene in the cell.

As used herein, “level of activity” is understood as the amount ofprotein activity, typically enzymatic activity, as determined by aquantitative, semi-quantitative, or qualitative assay. Activity istypically determined by monitoring the amount of product produced in anassay using a substrate that produces a readily detectable product,e.g., colored product, fluorescent product, or radioactive product.

As used herein, “changed as compared to a control” sample or subject isunderstood as having a level of the analyte or diagnostic or therapeuticindicator (e.g., marker) to be detected at a level that is statisticallydifferent than a sample from a normal, untreated, or control samplecontrol samples include, for example, cells in culture, one or morelaboratory test animals, or one or more human subjects. Methods toselect and test control samples are within the ability of those in theart. An analyte can be a naturally occurring substance that ischaracteristically expressed or produced by the cell or organism (e.g.,an antibody, a protein) or a substance produced by a reporter construct(e.g., β-galactosidase or luciferase). Depending on the method used fordetection the amount and measurement of the change can vary. Changed ascompared to a control reference sample can also include a change in oneor more signs or symptoms associated with or diagnostic of disease,e.g., cancer. Determination of statistical significance is within theability of those skilled in the art, e.g., the number of standarddeviations from the mean that constitute a positive result.

“Elevated” or “lower” refers to a patient's value of a marker relativeto the upper limit of normal (“ULN”) or the lower limit of normal(“LLN”) which are based on historical normal control samples. As thelevel of the marker present in the subject will be a result of thedisease, and not a result of treatment, typically a control sampleobtained from the patient prior to onset of the disease will not likelybe available. Because different labs may have different absoluteresults, values are presented relative to that lab's upper limit ofnormal value (ULN).

The “normal” level of expression of a marker is the level of expressionof the marker in cells of a subject or patient not afflicted withcancer. In one embodiment, a “normal” level of expression refers to thelevel of expression of the marker under normoxic conditions.

An “over-expression” or “high level of expression” of a marker refers toan expression level in a test sample that is greater than the standarderror of the assay employed to assess expression, and is preferably atleast 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 4, 5, 6, 7, 8, 9, or 10 times theexpression level of the marker in a control sample (e.g., sample from ahealthy subject not having the marker associated disease, i.e., cancer).In one embodiment, expression of a marker is compared to an averageexpression level of the marker in several control samples.

A “low level of expression” or “under-expression” of a marker refers toan expression level in a test sample that is less than at least 0.9,0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 times the expression level ofthe marker in a control sample (e.g., sample from a healthy subject nothaving the marker associated disease, i.e., cancer). In one embodiment,expression of a marker is compared to an average expression level of themarker in several control samples.

As used herein, “binding” is understood as having at least a 10² ormore, 10³ or more, preferably 10⁴ or more, preferably 10⁵ or more,preferably 10⁶ or more preference for binding to a specific bindingpartner as compared to a non-specific binding partner (e.g., binding anantigen to a sample known to contain the cognate antibody).

“Determining” as used herein is understood as performing an assay orusing a diagnostic method to ascertain the state of someone orsomething, e.g., the presence, absence, level, or degree of a certaincondition, biomarker, disease state, or physiological condition.

“Prescribing” as used herein is understood as indicating a specificagent or agents for administration to a subject.

As used herein, the terms “respond” or “response” are understood ashaving a positive response to treatment with a therapeutic agent,wherein a positive response is understood as having a decrease in atleast one sign or symptom of a disease or condition (e.g., tumorshrinkage, decrease in tumor burden, inhibition or decrease ofmetastasis, improving quality of life (“QOL”), delay of time toprogression (“TTP”), increase of overall survival (“OS”), etc.), orslowing or stopping of disease progression (e.g., halting tumor growthor metastasis, or slowing the rate of tumor growth or metastasis). Aresponse can also include an improvement in quality of life, or anincrease in survival time or progression free survival.

The terms “administer,” “administering” or “administration” can includeany method of delivery of a pharmaceutical composition or agent into asubject's system or to a particular region in or on a subject. Incertain embodiments of the invention, an Hsp90 inhibitor is administeredintravenously, intramuscularly, subcutaneously, intradermally,intranasally, orally, transcutaneously, or mucosally. In a preferredembodiment, an agent is administered intravenously. Administering can beperformed by a number of people working in concert. Administering anagent includes, for example, prescribing an agent to be administered toa subject and/or providing instructions, directly or through another, totake a specific agent, either by self-delivery, e.g., as by oraldelivery, subcutaneous delivery, intravenous delivery through a centralline, etc.; or for delivery by a trained professional, e.g., intravenousdelivery, intramuscular delivery, intratumoral delivery, etc.

As used herein, the term “high concentration” refers to theconcentration of SDC-TRAP that accumulates in target cells of theinvention due to the selective binding of the binding moiety of theSDC-TRAP to the target protein. In one embodiment, the concentration ishigher than in similar cells that do not overexpress the target protein,e.g., lung cancer cells as compared to non-cancerous lung cells. Inanother embodiment, the concentration is higher in target cells comparedto cells that do not express, or overexpress, the target protein. Inexemplary embodiments, the high concentration is 1.5, 2, 3, 4, 5, 10,15, 20, 50, 100, 1000 times or more than cells that are not targeted bythe SDC-TRAP molecules of the invention.

The term “moiety” refers generally to a portion of a molecule, which maybe a functional group, a set of functional groups, and/or a specificgroup of atoms within a molecule, that is responsible for acharacteristic chemical, biological, and/or medicinal property of themolecule.

The term “binding moiety” refers to low molecular weight (e.g., lessthan about 800, 700, 600, 500, 400, 300, 200, or 100 etc. Dalton)organic compounds, which may serve as a therapeutic or a regulator of abiological process. Binding moieties include molecules that can bind toa biopolymer such as protein, nucleic acid, or polysaccharide and actsas an effector, altering the activity or function of the biopolymer.Binding moieties can have a variety of biological functions, serving ascell signaling molecules, as tools in molecular biology, as drugs inmedicine, as pesticides in farming, and in many other roles. Thesecompounds can be natural (such as secondary metabolites) or artificial(such as antiviral drugs); they may have a beneficial effect against adisease (such as drugs) or may be detrimental (such as teratogens andcarcinogens). Biopolymers such as nucleic acids, proteins, andpolysaccharides (such as starch or cellulose) are not binding moieties,although their constituent monomers—ribo- or deoxyribo-nucleotides,amino acids, and monosaccharides, respectively—are often considered tobe. Small oligomers are also usually considered binding moieties, suchas dinucleotides, peptides such as the antioxidant glutathione, anddisaccharides such as sucrose.

As used herein, a “protein interacting binding moiety” or “bindingmoiety” refers to a binding moiety, or portion thereof, that interactswith a predetermined target. The interaction is achieved through somedegree of specificity and/or affinity for the target. Both specificityand affinity is generally desirable, although in certain cases higherspecificity may compensate for lower affinity and higher affinity maycompensate for lower specificity. Affinity and specificity requirementswill vary depending upon various factors including, but not limited to,absolute concentration of the target, relative concentration of thetarget (e.g., in cancer vs. normal cells), potency and toxicity, routeof administration, and/or diffusion or transport into a target cell. Thetarget can be a molecule of interest and/or localized in an area ofinterest. For example, the target can be a therapeutic target and/orlocalized in an area targeted for a therapy (e.g., a protein that isoverexpressed in cancerous cells, as compared to normal cells). In oneparticular example, a target can be a chaperonin protein such as Hsp90and the binding moiety can be an Hsp90 binding moiety (e.g.,therapeutic, cytotoxic, or imaging moiety). Preferentially, the bindingmoiety will enhance, be compatible with, or not substantially reduce,passive transport of a conjugate including the binding moiety into acell, e.g., a cell comprising a target protein.

The term “effector moiety” refers to a molecule, or portion thereof,that has an effect on a target and/or proximally to the target. Invarious preferred embodiments, the effector moiety is a binding moiety,or portion thereof. An effect can include, but is not limited to, atherapeutic effect, an imaging effect, and/or a cytotoxic effect. At amolecular or cellular level, an effect can include, but is not limitedto, promotion or inhibition of the target's activity, labeling of thetarget, and/or cell death. Preferentially, the effector moiety willenhance, be compatible with, or not substantially reduce, passivetransport of a conjugate including the effector moiety into a cellcomprising a target. Different effector moieties can be used togetherand therapeutics in accordance with the present invention may includemore than one effector moiety (e.g., two or more different (or same)effector moieties in a single therapeutic in accordance with the presentinvention, two or more different therapeutics in accordance with thepresent invention including different effector moieties).

In some embodiments, the effector moiety is selected from the groupconsisting of peptidyl-prolyl isomerase ligands; rapamycin, cyclosporinA; steroid hormone receptor ligands, antimitotic agents, actin bindingagents, camptothecins, topotecan, combretastatins, capecitabine,gemcitabine, vinca alkaloids, platinum-containing compounds, metformin,HDAC inhibitors, thymidylate synthase inhibitors; nitrogen mustards;5-fluorouracil (5-FU) and its derivatives, or a combination thereof.

In some embodiments, the effector moiety is selected from the groupconsisting of FK506; rapamycin, cyclosporin A, estrogen, progestin,testosterone, taxanes, colchicine, colcemid, nocadozole, vinblastine,vincristine, cytochalasin, latrunculin, phalloidin, lenalidomide,pomalidomide, SN-38, topotecan, combretastatins, capecitabine,gemcitabine, vinca alkaloids, metformin, suberoylanilidehydroxamic acid(SAHA), methotrexate, pemetrexed, raltitrexed, bendamustine, melphalan;5-fluorouracil (5-FU), vedotin and DM1, or a combination thereof.

The term “small molecule drug conjugate that is trapped intracellularly”or “binding moiety drug conjugate that is trapped intracellularly” or“SDC-TRAP” refers to a binding moiety and effector moiety joined to oneanother, or acting as if joined to one another. A binding moiety andeffector moiety can be joined through essentially any chemical orphysical force, either directly (e.g., binding moiety and effectormoiety viewed as two moieties on the same molecule, or a single moietyhaving both functions) or through an intermediate (e.g., linker). Forexample, a binding moiety and effector moiety can be joined by one ormore covalent bonds, ionic bonds, hydrogen bonds, the hydrophobiceffect, dipole-dipole forces, ion-dipole forces, dipole-induced dipoleforces, instantaneous dipole-induced dipole forces, and/or combinationsthereof. Preferentially, the SDC-TRAP will be capable of passive and/oractive transport into a cell comprising a target. Moreover, SDC-TRAPmolecules of the invention may comprise multiple effector moleculesconjugated to the binding moiety.

The term “linker” or “linking moiety,” as used herein in the context ofbinding moiety, effector moieties, and/or SDC-TRAPs refers to a chemicalmoiety that joins two other moieties (e.g., a binding moiety and aneffector moiety). A linker can covalently join a binding moiety and aneffector moiety. A linker can include a cleavable linker, for example anenzymatically cleavable linker. A linker can include a disulfide,carbamate, amide, ester, and/or ether linkers.

As used herein, a “ligand” is a substance (e.g., a binding moiety) thatcan form a complex with a biomolecule. The ligand and/or formation ofthe ligand-biomolecule complex can have a biological or chemical effect,such as a therapeutic effect, cytotoxic effect, and/or imaging effect.

As used herein, a “prodrug” is a pharmacological substance that isadministered in an inactive or less than fully active form and that issubsequently converted to an active pharmacological agent (i.e., thedrug) through a metabolic processes. Prodrugs can be used to improve howthe intended drug is absorbed, distributed, metabolized, and/orexcreted. A prodrug may also be used to improve how selectively theintended drug interacts with cells or processes that are not itsintended target (e.g., to reduce adverse or unintended effects of theintended drug, for example a chemotherapy drug).

The phrase “Hsp90 ligand or a prodrug thereof” refers generally tomolecules that bind to and in some cases effect Hsp90, and inactiveforms (i.e., prodrugs) thereof. An Hsp90 ligand can be an “Hsp90inhibitor,” which is understood as a therapeutic agent that reduces theactivity of Hsp90 either by directly interacting with Hsp90 or by, forexample, preventing the formation of the Hsp90/CDC37 complex such thatthe expression and proper folding of at least one client protein ofHsp90 is inhibited. “Hsp90” includes each member of the family of heatshock proteins having a mass of about 90-kilodaltons. For example, inhumans the highly conserved Hsp90 family includes cytosolic Hsp90^(α)and Hsp90^(β) isoforms, as well as GRP94, which is found in theendoplasmic reticulum, and HSP75/TRAP1, which is found in themitochondrial matrix. As used herein, Hsp90 inhibitors include, but arenot limited to ganetespib, geldanamycin (tanespimycin), e.g., IPI-493,macbecins, tripterins, tanespimycins, e.g., 17-AAG (alvespimycin),KF-55823, radicicols, KF-58333, KF-58332, 17-DMAG, IPI-504, BIIB-021,BBB-028, PU-H64, PU-H71, PU-DZ8, PU-HZ151, SNX-2112, SNX-2321, SNX-5422,SNX-7081, SNX-8891, SNX-0723, SAR-567530, ABI-287, ABI-328, AT-13387,NSC-113497, PF-3823863, PF-4470296, EC-102, EC-154, ARQ-250-RP, BC-274,VER-50589, KW-2478, BHI-001, AUY-922, EMD-614684, EMD-683671, XL-888,VER-51047, KOS-2484, KOS-2539, CUDC-305, WC-3100, CH-5164840, PU-DZ13,PU-HZ151, PU-DZ13, VER-82576, VER-82160, VER-82576, VER-82160,NXD-30001, NVP-HSP990, SST-0201CL1, SST-0115AA1, SST-0221AA1,SST-0223AA1, novobiocin (a C-terminal Hsp90i, herbinmycin A, radicicol,CCT018059, PU-H71, or celastrol.

The term “therapeutic moiety” refers to molecule, compound, or fragmentthereof that is used for the treatment of a disease or for improving thewell-being of an organism or that otherwise exhibit healing power (e.g.,pharmaceuticals, drugs, and the like). A therapeutic moiety can be achemical, or fragment thereof, of natural or synthetic origin used forits specific action against disease, for example cancer. Therapeuticagents used for treating cancer may be called chemotherapeutic agents.As described herein, a therapeutic moiety is preferentially a smallmolecule. Exemplary small molecule therapeutics include those that areless than 800 Daltons, 700 Daltons, 600 Daltons, 500 Daltons, 400Daltons, or 300 Daltons.

The term “cytotoxic moiety” refers to molecule, compound, or fragmentthereof that has a toxic or poisonous effect on cells, or that killscells. Chemotherapy and radiotherapy are forms of cytotoxic therapy.Treating cells with a cytotoxic moiety can produce a variety ofresults—cells may undergo necrosis, stop actively growing and dividing,or activate a genetic program of controlled cell death (i.e.,apoptosis). Examples of cytotoxic moieties include, but are not limitedto, SN-38, bendamustine, a vascular disrupting agent (VDA), doxorubicin,pemetrexed, vorinostat, lenalidomide, irinotecan, ganetespib, docetaxel,17-AAG, 5-FU, abiraterone, crizotinib, KW-2189, BUMB2, DC1, CC-1065,adozelesin, fulvestrant, topotecan or (a) fragment(s) thereof.

The term “imaging moiety” refers to a molecule, compound, or fragmentthereof that facilitates a technique and/or process used to createimages or take measurements of a cell, tissue, and/or organism (or partsor functions thereof) for clinical and/or research purposes. An imagingmoiety can produce, for example, a signal through emission and/orinteraction with electromagnetic, nuclear, and/or mechanical (e.g.,acoustic as in ultrasound) energy. An imaging moiety can be used, forexample, in various radiology, nuclear medicine, endoscopy,thermography, photography, spectroscopy, and microscopy methods.

“Pharmaceutical conjugate” refers to a non-naturally occurring moleculethat includes a binding moiety (e.g., an Hsp90-targeting moiety)associated with an effector moiety, where these two components may alsobe covalently bonded to each other either directly or through a linkinggroup.

The term “drug” refers to any active agent that affects any biologicalprocess. Active agents that are considered drugs for purposes of thisapplication are agents that exhibit a pharmacological activity. Examplesof drugs include active agents that are used in the prevention,diagnosis, alleviation, treatment or cure of a disease condition.

By “pharmacologic activity” is meant an activity that modulates oralters a biological process so as to result in a phenotypic change,e.g., cell death, cell proliferation etc.

By “pharmacokinetic property” is meant a parameter that describes thedisposition of an active agent in an organism or host.

By “half-life” is meant the time for one-half of an administered drug tobe eliminated through biological processes, e.g., metabolism, excretion,etc.

The term “efficacy” refers to the effectiveness of a particular activeagent for its intended purpose, i.e., the ability of a given activeagent to cause its desired pharmacologic effect.

Binding Moiety-Effector Moiety Drug Conjugates that are TrappedIntracellularly (SDC-TRAPs)

The present invention provides SDC-TRAPs, as well as SDC-TRAPcompositions, kits, and methods of use thereof. SDC-TRAPs include abinding moiety (e.g., a binding moiety such as a ligand) conjugated toan effector moiety (e.g., a pharmacological agent such as a drug orimaging agent). These two moieties can be joined by a linker, e.g., acovalently-bonded linking group. SDC-TRAPs are useful in a variety oftherapeutic, imaging, diagnostic, and/or research applications. In oneillustrative example of cancer therapy, an SDC-TRAP can be apharmaceutical conjugate of an Hsp90-binding moiety such as an Hsp90ligand or inhibitor associated with an effector moiety such as atherapeutic or cytotoxic agent.

In various embodiments, an SDC-TRAP can be further characterized in thatthe binding moiety (e.g., targeting moiety) and effector moiety aredifferent, such that the pharmaceutical conjugate may be viewed as aheterodimeric compound produced by the joining of two differentmoieties. In terms of function, SDC-TRAP molecules have a targetingfunctionality and effector functionality (e.g., therapeutic, imaging,diagnostic). These functions are provided by corresponding chemicalmoieties that can be different (or, in some cases, the same). SDC-TRAPscan include any one or more binding moieties conjugated to any one ormore effector moieties. In some embodiments, a composition or method caninclude a combination of two or more binding moeities and/or two or moreeffector moieties (e.g., a combination therapy and/or multi targettherapy) embodied in one or more different types of SDC-TRAPs.

In various embodiments, an SDC-TRAP is further characterized by itsability to passively diffuse and/or be actively transported into atarget cell of interest. The diffusion and/or transport properties ofthe SDC-TRAP can be derived, at least in part, from ionic, polar, and/orhydrophobic properties of the SDC-TRAP. In preferred embodiments, theSDC-TRAP enter cells primarily by passive diffusion. The diffusionand/or transport properties of the SDC-TRAP can be derived, at least inpart, from the molecular weight of the SDC-TRAP, the binding moiety, theeffector moiety, and/or the similarity in weight between the bindingmoiety and the effector moiety. SDC-TRAPs are desirably small, such asin comparison to antibody-drug conjugates (“ADCs”). For example, themolecular weight of an SDC-TRAP can be less than about 1600, 1500, 1400,1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, or 400 Daltons. Abinding moiety and an effector moiety can each be less than about 1000,900, 800, 700, 600, 500, 400, 300, or 200 Daltons. A binding moiety andan effector moiety can be approximately equal in size (e.g., differ inweight by less than 400, 350, 300, 250, 200, 150, 100, or 50 Daltons).

Delivery of an effector molecule by an SDC-TRAP can result in greaterpotency compared to administering an untargeted drug comprising the sameeffector moiety, for example, because the SDC-TRAP can be localized at adesired target for an extended period of time through the association ofa binding moiety and its target. Such localization can cause an effectormoiety to be active and/or released in a target cell and/or tissue overan extended period of time. This resonance time can be selected throughdeliberate design of a linker moiety. In contrast, administration of thedrug by itself in vivo can be more apt to have a shorter resonance timein a given target cell and/or tissue—if it traverses into the cell atall—due to the lack of an “anchor” within the cell.

SDC-TRAPs, in part because they comprise a targeting moiety and arerelatively small in size, can be efficiently taken up or internalized bya target cell. Conversely, uptake or internalization is relativelyinefficient for ADCs, which must deal with limited antigen expressionand relatively inefficient internalization mechanisms for the antibodyportion of the molecule. Hsp90 provides a good illustrative example of adifference between SDC-TRAPs and conventional ADCs. By way ofcomparison, the localization rate of radiolabeled monoclonal antibodiesat a tumor in patients is low, on the order of 0.003-0.08% of theinjected dose/g tumor. In contrast, a much higher accumulation rate(15-20% injected dose/g tumor) has been measured for SDC-TRAPs in mousetumor xenografts.

SDC-TRAP pharmaceutical conjugates in accordance with the presentinvention can represent a significant advance over the state of the artin targeted drugs. SDC-TRAPs have broad application in many therapeutic,imaging, and diagnostic application. As discussed above, SDC-TRAPs areadvantageously small in comparison to ADCs, enabling better penetrationof solid tumors and more rapid clearance from normal tissues (e.g.,reduced toxicity). The design of SDC-TRAPs (e.g., a structure-propertyrelationship) can be established using methods and rationales within thegrasp of those of ordinary skill in the art, and companion imagingdiagnostics for targeted therapies may also easily be provided, in viewof the simpler chemistry involved.

SDC-TRAPs of the invention are characterized by selective targeting ofSDC-TRAPs to target cells in which a target protein is overexpressed.This leads to high intracellular concentrations of SDC-TRAP molecules intarget cells as compared to non-targeted cells. Likewise, SDC-TRAPs ofthe invention are characterized by low concentrations of SDC-TRAP innon-targeted cells.

One illustrative embodiment involves a conjugate of an Hsp90 bindingmoiety linked to a chelator (i.e., the effector moiety, for metals suchas In or Gd, which conjugate may function as an imaging agent for thecells/tissues targeted by the conjugate). Another, illustrativeembodiment involves a conjugate of an Hsp90 binding moiety linked to achemotherapeutic (i.e., the effector moiety, for example, SN-38).Alternatively, an illustrative SDC-TRAP is contemplated wherein an Hsp90targeting moiety bearing radiolabeled halogen (e.g., such as an iodineisotope) can serve to image the cells/tissues targeted by the conjugate,and the effector moiety can be drug to treat the targeted cells/tissues.The progression of treatment may therefore be determined by imaging thetissues being treated and reviewing the images for the presence orabsence of the labeled conjugate. Such embodiments are readily adaptableto essentially any cancer, or other chemotherapeutic target. Moleculartargets (e.g., interacting with a binding moiety) used to target aparticular cell or tissue can be selected based upon their presence inthe target cell or tissue and/or their relative abundance in the targetcell or tissue (e.g., disease-related versus normal cells).

SDC-TRAP molecules of the present invention represent a new class ofdrugs. One particular advantage of SDC-TRAPs is that they can bedesigned to selectively deliver an effector moiety (e.g., achemotherapeutic drug) into a targeted cell because of the relativeoverexpression or presence of a binding moiety's molecular target in thecell. After the binding moiety binds the molecular target, the effectormoiety is thereafter available (e.g., through cleavage of a linkermoiety joining the binding moiety and the effector moiety) to act uponthe cell. Accordingly, SDC-TRAPs employ a different mechanism fromstrategies currently used in the art, for example delivering an Hsp90inhibitor to a cell using HPMA copolymer-Hsp90i conjugates, Hsp90iprodrugs, nanoparticle-Hsp90i conjugates, or micellar methodologies.

SDC-TRAPs can also described by the formula:

Binding moiety-L-E

where “binding moiety” is a protein interacting binding moiety; L is aconjugation or linking moiety (e.g., a bond or a linking group); and Eis an effector moiety. These elements are discussed in the context ofadditional illustrative examples below. However, while features of eachelement may be discussed separately, design and selection of an SDC-TRAPcan involve the interplay and/or cumulative effect of features of eachelement (e.g., diffusion, binding, and effect).

Once SDC-TRAP molecules of the invention enter a target cell theeffector molecule is released from the SDC-TRAP. In one embodiment, theeffector molecule has no activity until it is released from theSDC-TRAP. Accordingly, once the SDC-TRAP molecules enter a target cellan equilibrium exists between free and bound SDC-TRAP molecules. In oneembodiment, the effector moiety is only released from the SDC-TRAP whenthe SDC-TRAP is not associated with the target protein. For example,when an SDC-TRAP molecule is not bound intracellular enzymes can accessthe linker region thereby freeing the effector moiety. Alternatively,when free SDC-TRAP molecules may be able to release effector moleculesthrough, for example, hydrolysis of the bond or linker that connects thebinding moiety and effector moiety.

Accordingly, the rate of effector molecule release and the amount ofeffector molecule released can be controlled by using binding moietiesthat bind to the target protein with different affinities. For example,binding moieties that bind to the target protein with lower affinitywill be free, resulting in higher concentrations of unboundintracellular SDC-TRAP, and thereby resulting in higher concentrationsof free effector molecule. Therefore, in at least one embodiment,irreversibly-binding binding moieties are incompatible with certainaspects of the invention, e.g., those embodiments where effectormolecule release is based on free intracellular SDC-TRAP molecules.

In one embodiment, SDC-TRAPs have favorable safety profiles, forexample, when compared to, for example, the binding moiety or effectormolecule alone. One reason for the increased safety profile is the rapidclearance of SDC-TRAP molecules that do not enter into a target cell.

A number of exemplary SDC-TRAP molecules are set forth in the examples.Specifically a number of Hsp90-specific SDC-TRAP molecules are describedand used to demonstrate the efficacy of SDC-TRAP molecules.

Binding Moieties

A primary role of a binding moiety is to ensure that the SDC-TRAPdelivers its payload—the effector moiety—to its target by binding to amolecular target in or on a target cell or tissue. In this respect, itis not necessary that the binding moiety also have an effect on thetarget (e.g., in the case of an Hsp90-targeting moiety, to inhibit Hsp90in the manner that Hsp90 is are known to do, that is, exhibitpharmacological activity or interfere with its function), but in someembodiments, the binding moiety does have an effect on the target.Accordingly, in various embodiments, an activity of the SDC-TRAP is duesolely to the effector moiety exerting a pharmacological effect on thetarget cell(s), which has been better facilitated by the pharmaceuticalconjugate targeting the target cell(s). In other embodiments, anactivity of the SDC-TRAP is due in part to the binding moiety—that is,the binding moiety can have an effect beyond targeting.

The molecular target of a binding moiety may or may not be part of acomplex or structure of a plurality of biological molecules, e.g.,lipids, where the complexes or structures may include lipoproteins,lipid bilayers, and the like. However, in many embodiments, themolecular target to which the binding moiety binds will be free (e.g.,cytoplasmic globular protein and/or not be part of a macromolecularassembly or aggregation). The present invention can exploit theselectively high presence of a molecular target in locations of highphysiological activity (e.g., Hsp90 in oncological processes). Forexample, where a drug target is an intracellular drug target, acorresponding molecular target (e.g., Hsp90) can be present in the cell.Likewise, where a drug target is an extracellular drug target, acorresponding molecular target (e.g., Hsp90) can be extracellular,proximal, or associated with the extracellular cell membrane of thetarget cell or tissue.

In various embodiments, a binding moiety can effect a target cell ortissue (e.g., in the case of an Hsp90-targeting moiety that in factinhibits Hsp90, for example, Hsp90i). In such embodiments, apharmacological activity of the binding moiety contributes to,complements, or augments, the pharmacological activity of the effectormoiety. Such embodiments go beyond the advantages combination therapies(e.g., a cancer combination therapy of Hsp90i and a second drug such asganetespib or crizotinib) by providing a therapy that can be carried outby administration of a single SDC-TRAP that realizes both the benefitsof the combination therapy and targeting. Other examples of suchSDC-TRAPs include conjugates of an Hsp90i (such as ganetespib) and asecond cancer drug such as docetaxel or paclitaxel (e.g., in NSCLC);BEZ235 (e.g., in melanoma, prostate and/or NSCLC); temsirolimus (e.g.,renal cell carcinoma (RCC), colon, breast and/or NSCLC); PLX4032 (e.g.,in melanoma); cisplatin (e.g., colon, breast cancer); AZD8055 (e.g., inNSCLC); and crizotinib (e.g., ALK⁺ NSCLC).

A range of pharmaceutical activities can be achieved by judiciousselection of a binding moiety and an effector moiety. For example, fortreating solid tumors, e.g., colon cancer, high continuous doses ofantimetabolites such as capecitabine or gemcitabine tend to be requiredin combination with other drugs. A conjugate having an Hsp90-targetingmoiety with lower binding affinity or inhibitory activity to Hsp90,e.g., as determined by a HER2 degradation assay, can be designed to meetthis need. Such a conjugate can comprise an effector moiety that is astrong, potent antimetabolite such as 5-FU, to afford a high dose of theconjugate that may be dosed relatively frequently. Such an approach notonly achieves the aim of providing a high dose of an antimetabolitefragment at the tumor, but also lowers the toxicity of administering thedrug on its own, owing to the plasma stability of SDC-TRAPs of theinvention, and the ability of the Hsp90-targeting moiety to deliver theantimetabolite to the desired cells or tissues.

In embodiments where solid tumors such as SCLC or colorectal cancer areto be treated with drugs such as topotecan or irinotecan, only low dosesof the drug may be dosed. Due to the very high intrinsic activity ofthese drugs, an SDC-TRAP should be designed to provide a low dose ofsuch drugs at the target tissue. In this scenario, for example, anHsp90-targeting moiety having a higher binding affinity or inhibitoryactivity to Hsp90 (e.g., as determined by a HER2 degradation assay) cansufficiently maintain the presence of the drug in the tissue at a veryhigh level, to ensure that enough of the drug reaches and is retained bythe desired target tissue due to the low dosing.

In various illustrative embodiments where a molecular target of abinding moiety is Hsp90, the binding moiety can be an Hsp90-targetingmoiety, for example a triazole/resorcinol-based compound that bindsHsp90, or a resorcinol amide-based compound that binds Hsp90, e.g.,ganetespib, AUY-922 or AT-13387. In another embodiment, the bindingmoiety may advantageously be an Hsp90-binding compound of formula (I):

whereinR¹ may be alkyl, aryl, halide, carboxamide or sulfonamide; R² may bealkyl, cycloalkyl, aryl or heteroaryl, wherein when R² is a 6 memberedaryl or heteroaryl, R² is substituted at the 3- and 4-positions relativeto the connection point on the triazole ring, through which a linker Lis attached; and R³ may be SH, OH, —CONHR⁴, aryl or heteroaryl, whereinwhen R³ is a 6 membered aryl or heteroaryl, R³ is substituted at the 3or 4 position.

In another embodiment, the binding moiety may advantageously be anHsp90-binding compound of formula (II):

whereinR¹ may be alkyl, aryl, halo, carboxamido, sulfonamido; and R² may beoptionally substituted alkyl, cycloalkyl, aryl or heteroaryl. Examplesof such compounds include5-(2,4-dihydroxy-5-isopropylphenyl)-N-(2-morpholinoethyl)-4-(4-(morpholinomethyl)phenyl)-4H-1,2,4-triazole-3-carboxamideand5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(4-methylpiperazin-1-yl)phenyl)-N-(2,2,2-trifluoroethyl)-4H-1,2,4-triazole-3-carboxamide.

In another embodiment, the binding moiety may advantageously be anHsp90-binding compound of formula (III):

whereinX, Y, and Z may independently be CH, N, O or S (with appropriatesubstitutions and satisfying the valency of the corresponding atoms andaromaticity of the ring); R¹ may be alkyl, aryl, halide, carboxamido orsulfonamido; R² may be substituted alkyl, cycloalkyl, aryl orheteroaryl, where a linker L is connected directly or to the extendedsubstitutions on these rings; R³ may be SH, OH, NR⁴R⁵ AND —CONHR⁶, towhich an effector moiety may be connected; R⁴ and R⁵ may independentlybe H, alkyl, aryl, or heteroaryl; and R⁶ may be alkyl, aryl, orheteroaryl, having a minimum of one functional group to which aneffector moiety may be connected. Examples of such compounds include

In another embodiment, the binding moiety may advantageously be anHsp90-binding compound of formula (IV):

whereinR¹ may be alkyl, aryl, halo, carboxamido or sulfonamido; R² and R³ areindependently C₁-C₅ hydrocarbyl groups optionally substituted with oneor more of hydroxy, halogen, C₁-C₂ alkoxy, amino, mono- and di-C₁-C₂alkylamino; 5- to 12-membered aryl or heteroaryl groups; or, R² and R³,taken together with the nitrogen atom to which they are attached, form a4- to 8-membered monocyclic heterocyclic group, of which up to 5 ringmembers are selected from O, N and S. Examples of such compounds include

In certain embodiments, to enhance the bioavailability or delivery ofthe pharmaceutical conjugate, the binding moiety may be a prodrug of theHsp90-binding compound. FIG. 1 shows how the illustrated Hsp90-targetingmoiety may be suitably modified at one or more positions to enhance thephysical, pharmacokinetic or pharmacodynamic properties of theconjugate.

Specific examples of suitable Hsp90-targeting moieties includegeldanamycins, e.g.,

macbecins, tripterins, tanespimycins, e.g.,

novobiocin (a C-terminal Hsp90i.) The selection of other Hsp90-targetingmoieties will be within the grasp of one of ordinary skill in the art.Likewise, the selection of binding moieties suitable for other moleculartargets and/or other applications will be within the ability of one ofordinary skill in the art.

Additionally Hsp90 targeting moieties can be used to construct SDC-TRAPmolecules for the treatment of inflammation. For example, bindingmoieties comprising the compounds shown in Tables 5, 6, and 7 of U.S.Patent Publication 2010/0280032, which is incorporated herein byreference in its entirety, or compounds of any formula therein, ortautomers, pharmaceutically acceptable salts, solvates, clathrates,hydrates, polymorphs or prodrugs thereof, inhibit the activity of Hsp90and, thereby cause the degradation of Hsp90 client proteins. Any ofthese compounds may be coupled to an effector molecule to form anSDC-TRAP. The glucocorticoid receptor is a client protein of Hsp90 andbinds to Hsp90 when it is in the conformation that is able to bindglucocorticoid ligands such as cortisol. Once a glucocorticoid binds toGR, the receptor disassociates with Hsp90 and translocates to thenucleus where it modulates gene expression to reduce inflammatoryresponses such as proinflammatory cytokine production. Thus,glucocorticoids may be given to patients in need of immunosuppressionand patients with inflammatory and autoimmune disorders. Unfortunately,although glucocorticoids are effective at relieving inflammation, theyhave a number of severe side effects including osteoporosis, musclewasting, hypertension, insulin resistance, truncal obesity and fatredistribution, and inhibition of wound repair. Inhibition of Hsp90causes changes in GR activity which results in reduction of inflammatoryresponses similar to those seen for glucocorticoids. However, since themechanism for reducing inflammation is different than that ofglucocorticoids, it is expected that some or all of the side effects ofglucocorticoid treatment will be reduced or eliminated.

Effector Moieties

An effector moiety can be any therapeutic or imaging agent that can beconjugated to a binding moiety and, in a thus conjugated state,delivered to a molecular target of the binding moiety. An effectormolecule can, in some cases, require a linking moiety for conjugation(e.g., cannot be directly conjugated to a binding moiety). Similarly, aneffector molecule can, in some cases, impede or reduce the ability ofthe binding moiety and/or SDC-TRAP to reach a target as long as theSDC-TRAP can still effect the target. However, in preferred embodiments,an effector moiety is readily conjugatable and may benefits delivery to,and effecting, of the target.

In various embodiments, an SDC-TRAP, via an effector moiety, can haveother ways of cell penetration than simple passive diffusion. Such anexample is an SDC-TRAP including an antifolate or fragments thereof(e.g., temozolamide, mitozolamide, nitrogen mustards, estramustine, orchloromethine) as the effector moiety. In this case, a conjugate of abinding moiety (e.g., Hsp90 inhibitor) with pemetrexed (or itsfolate-recognizing fragment) can undergo folate receptor mediatedendocytosis rather than passive diffusion. Once in a target cell, theSDC-TRAP can bind the molecular target (e.g., Hsp90 protein) via itsbinding moiety (e.g., Hsp90 inhibitor).

As described in greater detail below, an effector moiety can comprise aregion that can be modified and/or participate in covalent linkage to abinding moiety without substantially adversely affecting the bindingmoiety's ability to bind to its target. An effector moiety can be apharmaceutical molecule or a derivative thereof, which essentiallyretains activity while conjugated to a binding moiety. It will beappreciated that drugs with otherwise good and desirable activity canprove challenging to administer conventionally (e.g., due to poorbioavailability or undesirable side-effects in vivo prior to reachingtheir target)—such drugs can be “reclaimed” for use as effector moietiesin the SDC-TRAPs of the present invention.

Examples of effector moieties include: peptidyl-prolyl isomeraseligands, e.g., FK506; rapamycin, cyclosporin A and the like; steroidhormone receptor ligands, e.g., naturally occurring steroid hormones,such as estrogen, progestin, testosterone, and the like, as well assynthetic derivatives and mimetics thereof; binding moieties that bindto cytoskeletal proteins, e.g., antimitotic agents, such as taxanes,colchicine, colcemid, nocadozole, vinblastine, and vincristine, actinbinding agents, such as cytochalasin, latrunculin, phalloidin, and thelike; lenalidomide, pomalidomide, camptothecins including

topotecan, combretastatins, capecitabine, gemcitabine, vinca alkaloids,platinum-containing compounds, metformin, HDAC inhibitors (e.g.,suberoylanilidehydroxamic acid (SAHA)), thymidylate synthase inhibitorssuch as methotrexate, pemetrexed, and raltitrexed; nitrogen mustardssuch as bendamustine and melphalan; 5-fluorouracil (5-FU) and itsderivatives; and agents used in ADC drugs, such as vedotin and DM1.

The effector moiety may be obtained from a library of naturallyoccurring or synthetic molecules, including a library of compoundsproduced through combinatorial means, i.e., a compound diversitycombinatorial library. When obtained from such libraries, the effectormoiety employed will have demonstrated some desirable activity in anappropriate screening assay for the activity. It is contemplated that inother embodiments, the pharmaceutical conjugate may include more thanone effector moiety(ies), providing the medicinal chemist with moreflexibility. The number of effector moieties linked to the bindingmoiety (e.g., Hsp90-targeting moiety) will generally only be limited bythe number of sites on the binding moiety (e.g., Hsp90-targeting moiety)and/or any linking moiety available for linking to an effector moiety;the steric considerations, e.g., the number of effector moieties thancan actually be linked to the binding moiety (e.g., Hsp90-targetingmoiety); and that the ability of the pharmaceutical conjugate to bind tothe molecular target (e.g., Hsp90 protein) is preserved. An example of atwo-effector moiety pharmaceutical conjugate can be seen in FIG. 2.

Specific drugs from which the effector moiety may be derived include:psychopharmacological agents, such as central nervous systemdepressants, e.g., general anesthetics (barbiturates, benzodiazepines,steroids, cyclohexanone derivatives, and miscellaneous agents),sedative-hypnotics (benzodiazepines, barbiturates, piperidinediones andtriones, quinazoline derivatives, carbamates, aldehydes and derivatives,amides, acyclic ureides, benzazepines and related drugs, phenothiazines,etc.), central voluntary muscle tone modifying drugs (anticonvulsants,such as hydantoins, barbiturates, oxazolidinediones, succinimides,acylureides, glutarimides, benzodiazepines, secondary and tertiaryalcohols, dibenzazepine derivatives, valproic acid and derivatives, GABAanalogs, etc.), analgesics (morphine and derivatives, oripavinederivatives, morphinan derivatives, phenylpiperidines,2,6-methane-3-benzazocaine derivatives, diphenylpropylamines andisosteres, salicylates, p-aminophenol derivatives, 5-pyrazolonederivatives, arylacetic acid derivatives, fenamates and isosteres, etc.)and antiemetics (anticholinergics, antihistamines, antidopaminergics,etc.); central nervous system stimulants, e.g., analeptics (respiratorystimulants, convulsant stimulants, psychomotor stimulants), narcoticantagonists (morphine derivatives, oripavine derivatives,2,6-methane-3-benzoxacine derivatives, morphinan derivatives)nootropics; psychopharmacological/psychotropics, e.g., anxiolyticsedatives (benzodiazepines, propanediol carbamates) antipsychotics(phenothiazine derivatives, thioxanthine derivatives, other tricycliccompounds, butyrophenone derivatives and isosteres, diphenylbutylaminederivatives, substituted benzamides, arylpiperazine derivatives, indolederivatives, etc.), antidepressants (tricyclic compounds, MAOinhibitors, etc.);

respiratory tract drugs, e.g., central antitussives (opium alkaloids andtheir derivatives); immunosuppressive agents; pharmacodynamic agents,such as peripheral nervous system drugs, e.g., local anesthetics (esterderivatives, amide derivatives); drugs acting at synaptic orneuroeffector junctional sites, e.g., cholinergic agents, cholinergicblocking agents, neuromuscular blocking agents, adrenergic agents,antiadrenergic agents; smooth muscle active drugs, e.g., spasmolytics(anticholinergics, musculotropic spasmolytics), vasodilators, smoothmuscle stimulants; histamines and antihistamines, e.g., histamine andderivative thereof (betazole), antihistamines (H₁-antagonists,H₂-antagonists), histamine metabolism drugs; cardiovascular drugs, e.g.,cardiotonics (plant extracts, butenolides, pentadienolids, alkaloidsfrom erythrophleum species, ionophores,-adrenoceptor stimulants, etc.),antiarrhythmic drugs, antihypertensive agents, antilipidemic agents(clofibric acid derivatives, nicotinic acid derivatives, hormones andanalogs, antibiotics, salicylic acid and derivatives), antivaricosedrugs, hemostyptics; chemotherapeutic agents, such as anti-infectiveagents, e.g., ectoparasiticides (chlorinated hydrocarbons, pyrethins,sulfurated compounds), anthelmintics, antiprotozoal agents, antimalarialagents, antiamebic agents, antileiscmanial drugs, antitrichomonalagents, antitrypanosomal agents, sulfonamides, antimycobacterial drugs,antiviral chemotherapeutics, etc., and cytostatics, i.e., antineoplasticagents or cytotoxic drugs, such as alkylating agents, e.g.,Mechlorethamine hydrochloride (Nitrogen Mustard, Mustargen, HN2),Cyclophosphamide (Cytovan, Endoxana), Ifosfamide (IFEX), Chlorambucil(Leukeran), Melphalan (Phenylalanine Mustard, L-sarcolysin, Alkeran,L-PAM), Busulfan (Myleran), Thiotepa (Triethylenethiophosphoramide),Carmustine (BiCNU, BCNU), Lomustine (CeeNU, CCNU), Streptozocin(Zanosar) and the like; plant alkaloids, e.g., Vincristine (Oncovin),Vinblastine (Velban, Velbe), Paclitaxel (Taxol), and the like;antimetabolites, e.g., Methotrexate (MTX), Mercaptopurine (Purinethol,6-MP), Thioguanine (6-TG), Fluorouracil (5-FU), Cytarabine (Cytosar-U,Ara-C), Azacitidine (Mylosar, 5-AZA) and the like; antibiotics, e.g.,Dactinomycin (Actinomycin D, Cosmegen), Doxorubicin (Adriamycin),Daunorubicin (duanomycin, Cerubidine), Idarubicin (Idamycin), Bleomycin(Blenoxane), Picamycin (Mithramycin, Mithracin), Mitomycin (Mutamycin)and the like, and other anticellular proliferative agents, e.g.,Hydroxyurea (Hydrea), Procarbazine (Mutalane), Dacarbazine (DTIC-Dome),Cisplatin (Platinol) Carboplatin (Paraplatin), Asparaginase (Elspar)Etoposide (VePesid, VP-16-213), Amsarcrine (AMSA, m-AMSA), Mitotane(Lysodren), Mitoxantrone (Novatrone), and the like;

anti-inflammatory agents; antibiotics, such as: aminoglycosides, e.g.,amikacin, apramycin, arbekacin, bambermycins, butirosin, dibekacin,dihydrostreptomycin, fortimicin, gentamicin, isepamicin, kanamycin,micronomcin, neomycin, netilmicin, paromycin, ribostamycin, sisomicin,spectinomycin, streptomycin, tobramycin, trospectomycin; amphenicols,e.g., azidamfenicol, chloramphenicol, florfenicol, and theimaphenicol;ansamycins, e.g., rifamide, rifampin, rifamycin, rifapentine, rifaximin;β-lactams, e.g., carbacephems, carbapenems, cephalosporins, cehpamycins,monobactams, oxaphems, penicillins; lincosamides, e.g., clinamycin,lincomycin; macrolides, e.g., clarithromycin, dirthromycin,erythromycin, etc.; polypeptides, e.g., amphomycin, bacitracin,capreomycin, etc.; tetracyclines, e.g., apicycline, chlortetracycline,clomocycline, etc.; synthetic antibacterial agents, such as2,4-diaminopyrimidines, nitrofurans, quinolones and analogs thereof,sulfonamides, sulfones;

antifungal agents, such as: polyenes, e.g., amphotericin B, candicidin,dermostatin, filipin, fungichromin, hachimycin, hamycin, lucensomycin,mepartricin, natamycin, nystatin, pecilocin, perimycin; syntheticantifungals, such as allylamines, e.g., butenafine, naftifine,terbinafine; imidazoles, e.g., bifonazole, butoconazole, chlordantoin,chlormidazole, etc., thiocarbamates, e.g., tolciclate, triazoles, e.g.,fluconazole, itraconazole, terconazole;

anthelmintics, such as: arecoline, aspidin, aspidinol, dichlorophene,embelin, kosin, napthalene, niclosamide, pelletierine, quinacrine,alantolactone, amocarzine, amoscanate, ascaridole, bephenium,bitoscanate, carbon tetrachloride, carvacrol, cyclobendazole,diethylcarbamazine, etc.;

antimalarials, such as: acedapsone, amodiaquin, arteether, artemether,artemisinin, artesunate, atovaquone, bebeerine, berberine, chirata,chlorguanide, chloroquine, chlorprogaunil, cinchona, cinchonidine,cinchonine, cycloguanil, gentiopicrin, halofantrine, hydroxychloroquine,mefloquine hydrochloride, 3-methylarsacetin, pamaquine, plasmocid,primaquine, pyrimethamine, quinacrine, quinidine, quinine, quinocide,quinoline, dibasic sodium arsenate; and

antiprotozoan agents, such as: acranil, tinidazole, ipronidazole,ethylstibamine, pentamidine, acetarsone, aminitrozole, anisomycin,nifuratel, tinidazole, benzidazole, suramin, and the like.

Conjugation and Linking Moieties

Binding moieties and effector moieties of the present invention can beconjugated, for example, through a linker or linking moiety L, where Lmay be either a bond or a linking group. For example, in variousembodiments, a binding moiety and an effector moiety are bound directlyor are parts of a single molecule. Alternatively, a linking moiety canprovide a covalent attachment between a binding moiety and effectormoiety. A linking moiety, as with a direct bond, can achieve a desiredstructural relationship between a binding moiety and effector moiety andor an SDC-TRAP and its molecular target. A linking moiety can be inert,for example, with respect to the targeting of a binding moiety andbiological activity of an effector moiety.

Appropriate linking moieties can be identified using the affinity,specificity, and/or selectivity assays described herein. Linkingmoieties can be selected based on size, for example, to provide anSDC-TRAP with size characteristics as described above. In variousembodiments, a linking moiety can be selected, or derived from, knownchemical linkers. Linking moieties can comprise a spacer groupterminated at either end with a reactive functionality capable ofcovalently bonding to the drug or ligand moieties. Spacer groups ofinterest include aliphatic and unsaturated hydrocarbon chains, spacerscontaining heteroatoms such as oxygen (ethers such as polyethyleneglycol) or nitrogen (polyamines), peptides, carbohydrates, cyclic oracyclic systems that may possibly contain heteroatoms. Spacer groups mayalso be comprised of ligands that bind to metals such that the presenceof a metal ion coordinates two or more ligands to form a complex.Specific spacer elements include: 1,4-diaminohexane, xylylenediamine,terephthalic acid, 3,6-dioxaoctanedioic acid,ethylenediamine-N,N-diacetic acid,1,1′-ethylenebis(5-oxo-3-pyrrolidinecarboxylic acid),4,4′-ethylenedipiperidine. Potential reactive functionalities includenucleophilic functional groups (amines, alcohols, thiols, hydrazides),electrophilic functional groups (aldehydes, esters, vinyl ketones,epoxides, isocyanates, maleimides), functional groups capable ofcycloaddition reactions, forming disulfide bonds, or binding to metals.Specific examples include primary and secondary amines, hydroxamicacids, N-hydroxysuccinimidyl esters, N-hydroxysuccinimidyl carbonates,oxycarbonylimidazoles, nitrophenylesters, trifluoroethyl esters,glycidyl ethers, vinylsulfones, and maleimides. Specific linkingmoieties that may find use in the SDC-TRAPs include disulfides andstable thioether moieties.

In various embodiments, a linking moiety is cleavable, for exampleenzymatically cleavable. A cleavable linker can be used to release aneffector moiety inside a target cell after the SDC-TRAP is internalized.The susceptibility of a linking moiety to cleavage can be used tocontrol delivery of an effector molecule. For example, a linking moietycan be selected to provide extended or prolonged release of an effectormoiety in a target cell over time (e.g., a carbamate linking moiety maybe subject to enzymatic cleavage by a carboxylesterase via the samecellular process used to cleave other carbamate prodrugs likecapecitabine or irinotecan). In these, and various other embodiments, alinking moiety can exhibit sufficient stability to ensure good targetspecificity and low systemic toxicity, but not so much stability that itresults in lowering the potency and efficacy of the SDC-TRAP.

Exemplary linkers are described in U.S. Pat. No. 6,214,345(Bristol-Myers Squibb), U.S. Pat. Appl. 2003/0096743 and U.S. Pat. Appl.2003/0130189 (both to Seattle Genetics), de Groot et al., J. Med. Chem.42, 5277 (1999); de Groot et al. J. Org. Chem. 43, 3093 (2000); de Grootet al., J. Med. Chem. 66, 8815, (2001); WO 02/083180 (Syntarga); Carl etal., J. Med. Chem. Lett. 24, 479, (1981); Dubowchik et al., Bioorg &Med. Chem. Lett. 8, 3347 (1998) and Doronina et al. BioConjug Chem.2006; Doronina et al. Nat Biotech 2003.

Identification and Selection of Targets and Corresponding SDC-TRAPs

The present invention provides for a broad class of pharmacologicalcompounds including an effector moiety conjugated to an binding moietydirecting the effector moiety to a biological target of interest. Whiletreating cancer using an Hsp90 inhibitor binding moiety conjugated to acytotoxic agent effector moiety is one illustrative example of thepresent invention, SDC-TRAPs are fundamentally broader in terms of theircompositions and uses.

In various embodiments, the broad class of SDC-TRAP pharmacologicalcompounds that are directed to biological targets have the followingproperties:

the biological target (a cell and/or tissue target of interest, e.g., atumor) should be effectible by an effector moiety, and the effectormoiety should be known or developed for the biological target (e.g.,chemotherapeutic agent for the tumor); the biological target should beassociated with a molecular target (e.g., biomolecule, capable of beingspecifically bound, that is uniquely represented in the biologicaltarget) that specifically interacts with a binding moiety, and thebinding moiety should be known or developed for the molecular target(e.g., ligand for the biomolecule); and the effector moiety and bindingmoiety should be amenable to coupling and should essentially retaintheir respective activity after coupling. Furthermore, the conjugateshould be capable of reaching and interacting with the molecular target,and in clinical applications should be suitable for administration to asubject (e.g., a subject can tolerate a therapeutically effective dose).

Examples of therapeutic molecular targets (i.e., binding moiety bindingpartners) for various conditions/disease states are presented in thetable below. A suitable binding moiety can be selected based upon agiven molecular target and/or a suitable effector moiety can be selectedbased upon a given condition/disease. In some cases, an FDA approvedtherapeutic agent can be used as an effector moiety (i.e., where the FDAapproved therapeutic agent is an effector moiety as described herein,for example, a binding moiety and not an antibody).

FDA Approved Condition/Disease State Molecular target(s) TherapeuticAgent Acute allograft rejection CD3E Muromonab (renal transplant)Acromegaly somatostatin receptor 1 Octreotide Actinic Keratosistoll-like receptor 7 Imiquimod Acute Coronary Syndrome P2Y12ADP-receptor Brilinta Acute Myocardial plasminogen Reteplase Infarctionalpha₁-proteinase inhibitor elastase, neutrophil expressed Alpha-1proteinase (A₁-PI) deficiency inhibitor Alzheimer's Disease BACE1Alzheimer's Disease soluble APP α and APP β Anemia erythropoietinreceptor Epoetin alfa Angina, chronic stable calcium channel,voltage-dependent, L type, alpha 1C Nicardipine subunit Angina, unstableP2Y12 ADP-receptor Brilinta Angioedema, hereditary kallikrein 1Ecallantide Angioedema, acute bradykinin B2 receptor Firazyr hereditaryAnkylosing spondylitis tumor necrosis factor Infliximab Anticoagulantserpin peptidase inhibitor, clade D (heparin cofactor), Ardeparin member1 (withdrawn) Arrhythmia (ventricular) potassium voltage-gated channel,subfamily H Propafenone (eag-related), member 2 Arrhythmia calciumchannel, voltage-dependent, P/Q type, alpha 1A Bepridil subunitArthritis/rheumatic dihydroorotate dehydrogenase (quinone) Leflunomidedisorders Arthritis/rheumatic interleukin 1 receptor, type I Anakinradisorders Asthma cysteinyl leukotriene receptor 1 Nedocromil Asthma IgEantibodies Omalizumab Atypical hemolytic uremic complement component 5Eculizumab syndrome (aHUS) Baldness steroid-5-alpha-reductase, alphapolypeptide 1 (3-oxo-5 Finasteride alpha-steroid delta 4-dehydrogenasealpha 1) Benign prostatic steroid-5-alpha-reductase, alpha polypeptide 1(3-oxo-5 Finasteride hyperplasia alpha-steroid delta 4-dehydrogenasealpha 1) Bone/vertebral fracture TGF-beta activated kinase 1/MAP3K7binding protein 2 — prevention Breast Cancer ER (estrogen receptor)Breast Cancer HER-2/neu Trastuzumab (HER-2) Breast Cancer tubulin, beta1 class VI Paclitaxel Breast Cancer chromodomain helicase DNA bindingprotein 1 Epirubicin Breast Cancer Tubulin Halaven Breast/Ovarian CancerBRCA genes Bronchitis, chronic phosphodiesterase 4 (PDE4) inhibitorsDaliresp Cardiac Ischemic integrin, beta 3 (platelet glycoprotein IIIa,antigen CD61) Abciximab Conditions Cancer CD74; Trop-2; CEACAM6 CancerEGFR Cardiovascular disease Matrix Mettaloproteinases Cardiovasculardisease VKORC1 Cardiovascular disease LDL Cervical Dystoniavesicle-associated membrane protein 1 (synaptobrevin 1) Botulinum toxintype B Chemoprotectant alkaline phosphatase, placental-like 2 AmifostineChonic myelogenous interferon (alpha, beta and omega) receptor 1Interferon alfa-2a leukemia Chronic Obstructive phosphodiesterase 4(PDE4) inhibitors Daliresp Pulmonary Disorder Chronic spasticity due toryanodine receptor 1 (skeletal) Dantrolene upper motor disorders ColonCancer guanylate cyclase 2C Colorectal Cancer EGFR Colorectal CancerKRAS Colorectal Cancer CEA Congestive Heart Failure B-type natriureticpeptide Congestive Heart Failure plasminogen Reteplase Crohn's Diseaseintegrin, alpha 4 (antigen CD49D, alpha 4 subunit of Natalizumab VLA-4receptor) Cryopyrin-associated interleukin 1, beta Canakinumab periodicsyndromes Cryopyrin-associated interleukin 1, alpha Rilonacept periodicsyndromes Depression 5HT1A receptor (a serotonin reuptake inhibitor)Viibryd Diabetes dipeptidyl peptidase-4 (DPP-4) enzyme TradjentaDiabetes protein kinase, AMP-activated, beta 1 non-catalytic subunitMetformin Diabetes amylase, alpha 2A (pancreatic) Acarbose Diabetesperoxisome proliferator-activated receptor gamma Troglitazone(withdrawn) Diabetes glucagon-like peptide 1 receptor Exenatide Diabetesreceptor (G protein-coupled) activity modifying protein 1 PramlintideDiabetes dipeptidyl-peptidase 4 Sitagliptin Edema potassiumvoltage-gated channel, Isk-related family, Indapamide member 1 Edemasolute carrier family 12 (sodium/potassium/chloride Bumetanidetransporters), member 2 Factor XIII (FXIII) enzyme replacement therapy(FactorXIII) Corifact deficiency, congenital Familial cold interleukin1, beta Canakinumab autoinflammatory syndrome Familial cold interleukin1, alpha Rilonacept autoinflammatory syndrome Gaucher Disease, type IUDP-glucose ceramide glucosyltransferase Miglustat GI stromal tumors(GIST), Bcr-Abl tyrosine kinase (an abnormal tyrosine kinase) metastaticmalignant Glaucoma prostaglandin F receptor (FP) LatanoprostGranulomatous disease, interferon gamma receptor 1 Interferon chronicgamma-1b Growth disorder insulin-like growth factor 1 receptorMecasermin Growth hormone deficiency growth hormone releasing hormonereceptor Sermorelin Hairy cell leukemia interferon (alpha, beta andomega) receptor 1 Interferon alfa-2a Hairy cell leukemia adenosinedeaminase Pentostatin Heartburn (Gastric reflux) 5-hydroxytryptamine(serotonin) receptor 4, G Cisapride protein-coupled (withdrawn)Hemophilia (prevent plasminogen activator, tissue Tranexamic acidbleeding) Hepatitis C interferon (alpha, beta and omega) receptor 1Interferon alfa-2a Hepatitis C (genotype 1) hepatitis C virusnon-structural protein 3 (NS3) serine Victrelis protease Hepatitis C(genotype 1) hepatitis C virus non-structural protein 3 (NS3)/4A serineIncivek protease Hepatocellular Carcinoma α-fetoprotein HIV chemokine(C-C motif) receptor 5 (gene/pseudogene) Maraviroc HIV HIV-1 reversetranscriptase Edurant Hyperammonemia carbamoyl-phosphate synthase 1,mitochondrial Carglumic acid Hypercalcemia in patients calcium-sensingreceptor Cinacalcet with parathyroid carcinoma Hypercholesterolemia3-hydroxy-3-methylglutaryl-CoA reductase Lovastatin Hyperlipidemia NPC1(Niemann-Pick disease, type C1, gene)-like 1 Ezetimibe Hyperplasiasteroid-5-alpha-reductase, alpha polypeptide 1 (3-oxo-5 Finasteridealpha-steroid delta 4-dehydrogenase alpha 1) Hypertension adrenoceptoralpha 1D Terazosin Hypertension calcium channel, voltage-dependent, P/Qtype, alpha 1A Bepridil subunit Hypertension calcium channel,voltage-dependent, N type, alpha 1B Amlodipine subunit Hypertensionangiotensin II receptor, type Losartan Hypertension renin AliskirenHypertension AT1 subtype angiotensin II receptor Edarbi Hypertensionmembrane metallo-endopeptidase Candoxatril Increase bone density,parathyroid hormone 1 receptor Teriparatide prevent bone fractureInfections, acute skin and penicillin-binding proteins Teflaro skinstructure Infections, bacterial dipeptidase 1 (renal) Cilastatin(adjuvant) Infections (bone marrow colony stimulating factor 3 receptor(granulocyte) Filgrastim transplant, etc.) Infections, colonystimulating factor 2 receptor, alpha, low-affinity Sargramostimimmunomodulatory agents (granulocyte-macrophage) Infertility folliclestimulating hormone receptor Urofollitropin Inflammation C ReactiveProtein Interstitial cystitis, bladder fibroblast growth factor 1(acidic) Pentosan pain/discomfort due to polysulfate Irritable BowelSyndrome chloride channel, voltage-sensitive 2 Lubiprostone Kaposi'ssacroma, interferon (alpha, beta and omega) receptor 1 Interferonalfa-2a AIDS-related Leukemia/Lymphoma CD20 Antigen Leukemia/LymphomaCD30 Leukemia/Lymphoma PML/RAR alpha Leukemia, chronic myeloidproto-oncogene tyrosine-protein kinase Src Dasatinib Leukemia, myeloidCD33, Myeloid cell surface antigen CD33 Gemtuzumab ozogamicin(withdrawn) Lipodystrophy human GRF receptors Egrifta Lung Cancer ALKLung Cancer CD98; fascin; 14-3-3 eta Lymphocytic leukemia, polymerase(DNA directed), alpha 1, catalytic subunit Fludarabine B-cell chronicLymphocytic leukemia, CD52 (CAMPATH-1 antigen precursor) AlemtuzumabB-cell chronic Lymphocytic leukemia, membrane-spanning 4-domains,subfamily A, member 1 Rituximab chronic Lymphoma, Hodgkin's chemokine(C-X-C motif) receptor 4 Plerixafor Lymphoma, Hodgkin's CD30 AdcetrisLymphoma, mantle cell proteasome (prosome, macropain) subunit, betatype, 1 Bortezomib Lymphoma, systemic CD30 Adcetris anaplastic largecell Lymphocytic leukemia, histone deacetylase 1 Vorinostat T-cellMelanoma S100 protein Melanoma, metastatic (with mutated form of BRAfthat facilitates cell growth Zelboraf BRAFV600E mutation) Melanoma,metastatic CTLA-4 Yervoy Migrane Headache carbonic anhydrase IITopiramate Muckle-Wells syndrome interleukin 1, beta CanakinumabMuckle-Wells syndrome interleukin 1, alpha Rilonacept Multiple Sclerosissphingosine-1-phosphate receptor 1 Fingolimod Myeloma, multiplechemokine (C-X-C motif) receptor 4 Plerixafor Myeloma, multipleproteasome (prosome, macropain) subunit, beta type, 1 BortezomibMyocardial Infarction Troponin I Myocardial Infarction, P2Y12ADP-receptor Brilinta non-ST-elevation Myocardial Infarction, P2Y12ADP-receptor Brilinta ST-elevation N-acetylglutamate synthasecarbamoyl-phosphate synthase 1, mitochondrial Carglumic acid (NAGS)deficiency Nausea/vomiting 5-hydroxytryptamine (serotonin) receptor 3A,ionotropic Ondansetron Nausea/vomiting tachykinin receptor 1 AprepitantNausea/vomiting (severe) cannabinoid receptor 1 (brain) MarinolNon-Hodgkin's Lymphoma membrane-spanning 4-domains, subfamily A, member1 Rituximab Non-small cell lung cancer phosphoribosylglycinamideformyltransferase, Pemetrexed phosphoribosylglycinamide synthetase,phosphoribosylaminoimidazole synthetase Non-small cell lung cancerepidermal growth factor receptor Gefitinib Non-small cell lung cancerthe ATP-binding pocket of target protein kinases Xalkori (that isALK-positive) Obesity lipase, gastric/pancreatic lipase Orlistat OvarianCancer IGF-II; leptin; osteopontin; prolactin Oral mucositis fibroblastgrowth factor receptor 2 Palifermin Organ rejection FK506 bindingprotein 1A, 12 kDa Tacrolimus prophylaxsis Organ rejection IMP (inosine5′-monophosphate) dehydrogenase 2 Mycophenolate prophylaxsis mofetilOrgan rejection interleukin 2 receptor, alpha Daclizumab prophylaxsisOrgan rejection FK506 binding protein 12-rapamycin associated protein 1Sirolimus prophylaxsis Organ rejection protein phosphatase 3, regulatorysubunit B, beta Cyclosporine prophylaxsis Organ rejection CD80 and CD86,blocks CD28 mediated costimulation of T Nulojix prophylaxsis lymphocytesOsteoporosis interferon gamma receptor 1 Interferon gamma-1bOsteoporosis (prophylaxsis) TGF-beta activated kinase 1/MAP3K7 bindingprotein 2 Denosumab Paget's Disease farnesyl diphosphate synthasePamidronate Pancreatic Cancer CA19-9 Parkinson's Diseasecatechol-O-methyltransferase Tolcapone (withdrawn) Parkinson's Diseasemonoamine oxidase B Selegiline Paroxysmal nocturnal complement component5 Eculizumab hemoglobinuria Pneumonia, susceptible penicillin-bindingproteins Teflaro bacterial community-acquired Poisoning, ethylene glycolalcohol dehydrogenase 1B (class I), beta polypeptide Fomepizole ormethanol Psoriasis, plaque interleukin 12B (natural killer cellstimulatory factor 2, Ustekinumab cytotoxic lymphocyte maturation factor2, p40) Psoriasis, plaque integrin, alpha L (antigen CD11A (p180),lymphocyte Efalizumab function-associated antigen 1; alpha polypeptide)(withdrawn) Psoriasis, chronic plaque T-cell surface antigen CD2precursor Alefacept Psoriatic Arthritis tumor necrosis factor InfliximabProstate Cancer PSA (prostate specific antigen) Prostate hyperplasia,benign adrenoceptor alpha 1D Terazosin Pulmonary embolism Factor XaXarelto Pulmonary hypertension endothelin receptor type B Bosentan Renalcell carcinoma v-raf-1 murine leukemia viral oncogene homolog 1Sorafenib Renal cell carcinoma fms-related tyrosine kinase 1 (vascularendothelial growth Sunitinib factor/vascular permeability factorreceptor) Renal cell carcinoma vascular endothelial growth factor ABevacizumab Rheumatoid arthritis TNF-α Rheumatoid arthritis IL-6Rheumatoid arthritis inhibitor of kappa light polypeptide gene enhancerin Auranofin B-cells, kinase beta Rheumatoid arthritis tumor necrosisfactor Infliximab Rheumatoid arthritis CD80 (T-lymphocyte activationantigen CD80) Abatacept Rheumatoid arthritis interleukin 6 receptorTocilizumab Rheumatoid arthritis CEP-1 Schizophrenia CYP2D6 Scorpionstings venom toxins Anascorp Seizures carbonic anhydrase II TopiramateSeizures solute carrier family 6 (neurotransmitter transporter,Tiagabine GABA), member 1 Seizures 4-aminobutyrate aminotransferaseDivalproex sodium Seizures Gamma-amino butyric acid (GABA) Sepsis,severe coagulation factor VIII (Factors Va and VIIIa), Drotrecogin alfaprocoagulant component Small Cell Lung Cancer topoisomerase (DNA) IIalpha 170 kDa Etoposide Small Cell Lung Cancer topoisomerase (DNA) ITopotecan Stroke thrombin Pradaxa Stroke Factor Xa Xarelto Stroke,thrombotic purinergic receptor P2Y, G-protein coupled, 12 TiclopidineSystemic embolism Factor Xa Xarelto systemic embolism in thrombinPradaxa non-valvular atrial fibrillation Systemic lupus human Blymphocyte stimulator protein (BLyS) Benlysta erythematosus TesticularCancer LDH Throid Cancer Metastasis Thyro-globulin Thrombocythemiaphosphodiesterase 4B, cAMP-specific Amrinone Thrombocytopeniamyeloproliferative leukemia virus oncogene expression Romiplostimproduct Thrombocytopenia interleukin 11 receptor, alpha OprelvekinThrombosis, Deep vein Factor Xa Xarelto Thyroid Cancer protein kinasesof the VEGF, EGFR, and/or RET pathways Caprelsa Tyrosinemia type I,4-hydroxyphenylpyruvate dioxygenase Nitisinone hereditary Ulcer(anti-ulcer agent) ATPase, H+/K+ exchanging, alpha polypeptideOmeprazole Ulcers, diabetic neuropathic platelet-derived growth factorreceptor, beta polypeptide Becaplermin Urothelial Cell Carcinoma BladderTumor Antigen

Examples of imaging/diagnostic molecular targets (i.e., binding moietybinding partners) for various conditions/disease states are presented inthe table below. A suitable binding moiety can be selected based upon agiven molecular target and/or a suitable effector moiety can be selectedbased upon a given condition/disease. In some cases, an FDA approvedimaging/diagnostic agent can be used as an effector moiety (i.e., wherethe FDA approved imaging/diagnostic agent is an effector moiety asdescribed herein, for example, a binding moiety and not an antibody).

FDA Approved Imaging/ Condition/Disease State Molecular target(s)Diagnostic Alzheimer's disease, stroke, cerebral blood flowschizophrenia (hemoglobin) Alzheimer's disease β-amyloid protein (can beused to monitor progression of the disease) Diagnostic (screening testpancreatic lipase Bentiromide for exocrine pancreatic insufficiency andto monitor the adequacy of supplemental pancreatic therapy) Diagnosticfor bone density parathyroid hormone 1 Teriparatide receptorDiagnostic/imaging proteasome (prosome, Capromab macropain) subunit,alpha type, 6 pseudogene 1 Diagnostic for MRI to Paramagneticmacrocyclic Gadavist visualize blood brain contrast agentbarrier/abnormal vascularity of the CNS (to diagnose disorders of thebrain and spine) General Cognitive Decline thining of the cerebral(Dementia, Alzheimer's cortex Disease, Parkinson's Disease, etc.)Inflammation/tumor (radiolabeled) 18F- progression fludeoxyglucoseOsteoarthritis cartilage (collagen and proteoglycan) degenerationParkinson's syndrome Dopamine receptors DaTscan (diagnostic that detectsdopamine receptors) Thyroid Cancer thyroid stimulating Thyrotropinhormone receptor alfa

Imaging Moieties, and Diagnostic and Research Applications

In various embodiments, the effector moiety is an imaging moiety—thatis, a molecule, compound, or fragment thereof that facilitates atechnique and/or process used to create images or take measurements of acell, tissue, and/or organism (or parts or functions thereof) forclinical and/or research purposes. An imaging moiety can produce, forexample, a signal through emission and/or interaction withelectromagnetic, nuclear, and/or mechanical (e.g., acoustic as inultrasound) energy. An imaging moiety can be used, for example, invarious radiology, nuclear medicine, endoscopy, thermography,photography, spectroscopy, and microscopy methods.

Imaging studies can be used, for example, in a clinical or researchsetting to diagnose a subject, select a subject for therapy, select asubject for participation in a clinical trial, monitor the progressionof a disease, monitor the effect of therapy, to determine if a subjectshould discontinue or continue therapy, to determine if a subject hasreached a clinical end point, and to determine recurrence of a disease.Imaging studies can be used, for example, to conduct research toidentify effective interacting moieties and/or effector moieties and/orcombinations thereof, to identify effective dosing and dose scheduling,to identify effective routes of administration, and to identify suitabletargets (e.g., diseases susceptible to particular treatment).

Methods of Making Pharmaceutical Conjugates

The pharmaceutical conjugates, i.e., SDC-TRAPs, of the invention may beprepared using any convenient methodology. In a rational approach, thepharmaceutical conjugates are constructed from their individualcomponents, binding moiety, in some cases a linker, and effector moiety.The components can be covalently bonded to one another throughfunctional groups, as is known in the art, where such functional groupsmay be present on the components or introduced onto the components usingone or more steps, e.g., oxidation reactions, reduction reactions,cleavage reactions and the like. Functional groups that may be used incovalently bonding the components together to produce the pharmaceuticalconjugate include: hydroxy, sulfhydryl, amino, and the like. Theparticular portion of the different components that are modified toprovide for covalent linkage will be chosen so as not to substantiallyadversely interfere with that components desired binding activity, e.g.,for the effector moiety, a region that does not affect the targetbinding activity will be modified, such that a sufficient amount of thedesired drug activity is preserved. Where necessary and/or desired,certain moieties on the components may be protected using blockinggroups, as is known in the art, see, e.g., Green & Wuts, ProtectiveGroups in Organic Synthesis (John Wiley & Sons) (1991).

Alternatively, the pharmaceutical conjugate can be produced using knowncombinatorial methods to produce large libraries of potentialpharmaceutical conjugates which may then be screened for identificationof a bifunctional, molecule with the pharmacokinetic profile.Alternatively, the pharmaceutical conjugate may be produced usingmedicinal chemistry and known structure-activity relationships for thetargeting moiety and the drug. In particular, this approach will provideinsight as to where to join the two moieties to the linker.

A number of exemplary methods for preparing SDC-TRAP molecules are setforth in the examples. As one of skill in the art will understand, theexemplary methods set forth in the examples can be modified to makeother SDC-TRAP molecules.

Methods of Use, Pharmaceutical Preparations, and Kits

The pharmaceutical conjugates find use in treatment of a host condition,e.g., a disease condition. In these methods, an effective amount of thepharmaceutical conjugate is administered to the host, where “effectiveamount” means a dosage sufficient to produce the desired result, e.g.,an improvement in a disease condition or the symptoms associatedtherewith. In many embodiments, the amount of drug in the form of thepharmaceutical conjugate that need be administered to the host in orderto be an effective amount will vary from that which must be administeredin free drug form. The difference in amounts may vary, and in manyembodiments may range from two-fold to ten-fold. In certain embodiments,e.g., where the resultant modulated pharmacokinetic property orproperties result(s) in enhanced activity as compared to the free drugcontrol, the amount of drug that is an effective amount is less than theamount of corresponding free drug that needs to be administered, wherethe amount may be two-fold, usually about four-fold and more usuallyabout ten-fold less than the amount of free drug that is administered.

The pharmaceutical conjugate may be administered to the host using anyconvenient means capable of producing the desired result. Thus, thepharmaceutical conjugate can be incorporated into a variety offormulations for therapeutic administration. More particularly, thepharmaceutical conjugate of the present invention can be formulated intopharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers or diluents, and may be formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants and aerosols. As such,administration of the pharmaceutical conjugate can be achieved invarious ways, including oral, buccal, rectal, parenteral,intraperitoneal, intradermal, transdermal, intracheal, etc.,administration. In pharmaceutical dosage forms, the pharmaceuticalconjugate may be administered alone or in combination with otherpharmaceutically active compounds.

The subject methods find use in the treatment of a variety of differentdisease conditions. In certain embodiments, of particular interest isthe use of the subject methods in disease conditions where an activeagent or drug having desired activity has been previously identified,but which active agent or drug does not bind to its target with desiredaffinity and/or specificity. With such active agents or drugs, thesubject methods can be used to enhance the binding affinity and/orspecificity of the agent for its target.

The specific disease conditions treatable by with the subjectbifunctional compounds are as varied as the types of drug moieties thatcan be present in the pharmaceutical conjugate. Thus, disease conditionsinclude cellular proliferative diseases, such as neoplastic diseases,autoimmune diseases, central nervous system or neurodegenerativediseases, cardiovascular diseases, hormonal abnormality diseases,infectious diseases, and the like.

By treatment is meant at least an amelioration of the symptomsassociated with the disease condition afflicting the host, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g., symptom, associated with thepathological condition being treated, such as inflammation and painassociated therewith. As such, treatment also includes situations wherethe pathological condition, or at least symptoms associated therewith,are completely inhibited, e.g., prevented from happening, or stopped,e.g., terminated, such that the host no longer suffers from thepathological condition, or at least the symptoms that characterize thepathological condition.

Methods of use of the invention extend beyond strict treatment of adisease. For example, the invention includes uses in a clinical orresearch setting to diagnose a subject, select a subject for therapy,select a subject for participation in a clinical trial, monitor theprogression of a disease, monitor the effect of therapy, to determine ifa subject should discontinue or continue therapy, to determine if asubject has reached a clinical end point, and to determine recurrence ofa disease. The invention also includes uses in conducting research toidentify effective interacting moieties and/or effector moieties and/orcombinations thereof, to identify effective dosing and dose scheduling,to identify effective routes of administration, and to identify suitabletargets (e.g., diseases susceptible to particular treatment).

A variety of hosts are treatable according to the subject methods.Generally such hosts are “mammals” or “mammalian,” where these terms areused broadly to describe organisms which are within the class Mammalia,including the orders carnivore (e.g., dogs and cats), rodentia (e.g.,mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees,and monkeys). In many embodiments, the hosts will be humans.

The invention provides kits for treating a subject in need thereofcomprising at least one SDC-TRAP and instruction for administering atherapeutically effective amount of the at least one SDC-TRAP to thesubject, thereby treating the subject. The invention also provides kitsfor imaging, diagnosing, and/or selecting a subject comprising at leastone SDC-TRAP and instruction for administering an effective amount of atleast one SDC-TRAP to the subject, thereby imaging, diagnosing, and/orselecting the subject.

Kits with unit doses of the pharmaceutical conjugate, usually in oral orinjectable doses and often in a storage stable formulation, areprovided. In such kits, in addition to the containers containing theunit doses, an informational package insert describing the use andattendant benefits of the drugs in treating pathological condition ofinterest will be included. Preferred compounds and unit doses are thosedescribed herein above.

The invention also provides methods for treatment of a disease ordisorder in which the subject to be treated is selected for treatmentbased on the presence of, or the overexpression of, a particularprotein. For example, subjects may be selected for treatment of cancerbased on the presence of greater the normal levels of Hsp90. In thiscase, subjects would be administered an SDC-TRAP that comprises abinding moiety that selectively binds to Hsp90.

The invention provides methods of treating or preventing an inflammatorydisorder in a subject, comprising administering to the subject aneffective amount of a compound represented by any one of formula (I)through (LXXII), or any embodiment thereof, or a compound shown in Table5, 6, or 7 as disclosed in U.S. Patent Publication 2010/0280032. In oneembodiment, the compound or binding moiety or SDC-TRAP may beadministered to a human to treat or prevent an inflammatory disorder. Inanother embodiment, the inflammatory disorder is selected from the groupconsisting of transplant rejection, skin graft rejection, arthritis,rheumatoid arthritis, osteoarthritis and bone diseases associated withincreased bone resorption; inflammatory bowel disease, ileitis,ulcerative colitis, Barrett's syndrome, Crohn's disease; asthma, adultrespiratory distress syndrome, chronic obstructive airway disease;corneal dystrophy, trachoma, onchocerciasis, uveitis, sympatheticophthalmitis, endophthalmitis; gingivitis, periodontitis; tuberculosis;leprosy; uremic complications, glomerulonephritis, nephrosis;sclerodermatitis, psoriasis, eczema; chronic demyelinating diseases ofthe nervous system, multiple sclerosis, AIDS-related neurodegeneration,Alzheimer's disease, infectious meningitis, encephalomyelitis,Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosisviral or autoimmune encephalitis; autoimmune disorders, immune-complexvasculitis, systemic lupus and erythematodes; systemic lupuserythematosus (SLE); cardiomyopathy, ischemic heart diseasehypercholesterolemia, atherosclerosis, preeclampsia; chronic liverfailure, brain and spinal cord trauma. In another embodiment, anSDC-TRAP, or a compound shown in Table 5, 6, or 7 as disclosed in U.S.Patent Publication 2010/0280032, is administered with an additionaltherapeutic agent. In another embodiment, the additional therapeuticagent may an anti-inflammatory agent.

In one embodiment, an SDC-TRAP that is administered to a subject butdoes not enter a target cell is rapidly cleared from the body. In thisembodiment, the SDC-TRAP that does not enter a target cell is rapidlycleared in order to reduce the toxicity due to the components of theSDC-TRAP, the degradation products of the SDC-TRAP or the SDC-TRAPmolecule. Clearance rate can be determined by measuring the plasmaconcentration of the SDC-TRAP molecule as a function of time.

Likewise, SDC-TRAP molecules that enter non-targeted cells by passivediffusion rapidly exit the non-targeted cell or tissue and are eithereliminated from the subject or proceed to enter and be retained atargeted cell or tissue. For example, an SDC-TRAP that is intended totreat tumor cells and is targeted to tumor cells that overexpress, forexample, Hsp90 will accumulate selectively in tumor cells thatoverexpress Hsp90. Accordingly, very low levels of this exemplarySDC-TRAP will be present in non-tumor tissue such as normal lung tissue,heart, kidney, and the like. In one embodiment, the safety of theSDC-TRAP molecules of the invention can be determined by their lack ofaccumulation in non-targeted tissue. Conversely, the safety of theSDC-TRAP molecules of the invention can be determined by their selectiveaccumulation in the targeted cells and/or tissue.

EXAMPLES

The following examples, which are briefly summarized and then discussedin turn below, are offered by way of illustration and not by way oflimitation.

Example 1 presents the synthesis of exemplary SDC-TRAPs.

Example 2 presents the targeted delivery of exemplary SDC-TRAPs.

Example 3 presents an exemplary assay for selecting binding moieties.

Example 4 presents the cytotoxicity of exemplary SDC-TRAPs.

Example 5 presents the stability of exemplary SDC-TRAPs in plasma.

Example 6 presents a detailed schematic for the synthesis of anexemplary SDC-TRAP.

Example 7 presents results of tests using the SDC-TRAP of Example 6.

Example 8 presents the synthesis and testing of a lenalidomide-basedSDC-TRAP.

Examples 9 and 10 present examples of IC₅₀ value determinations.

Example 11 presents an exemplary Hsp90α binding assay.

Example 12 presents an exemplary HER2 degradation assay.

Example 13 presents an exemplary cytotoxicity assay.

Example 14 presents an exemplary plasma stability protocol.

Example 15 presents an exemplary tissue distribution extractionprocedure.

Example 16 presents an exemplary tissue distribution study.

Examples 17 and 18 present examples of SDC-TRAP stability in mouseplasma and cell culture media.

Examples 19-29 present synthesis and IC₅₀ data for different exemplarySDC-TRAPs. Within examples 19-29, exemplary synthetic schemes are setforth. It is to be understood that the additional exemplary compoundswere synthesized according to the methods described for the exemplarysynthetic schemes.

Example 30 sets forth the identification and use of SDC-TRAPs forprevention and treatment of chronic bronchitis and asthma.

Example 31 sets forth the identification and use of SDC-TRAPs forprevention and treatment of skin cancers and actinic keratosis.

Example 1

SDC-TRAPs of an exemplary embodiment may be prepared in the followingmanner:

The synthesis of Compound 1 and Compound 3 are discussed in WO2007/139968 and WO 2004/012661, respectively.

Synthesis of Compound 2 (STEP-1): To a solution of 1.0 g (2.48 mmols) ofCompound 1 in 60 mL of 1:1:1-Methanol:Tetrahydrofuran:Acetic acid wasadded 75 mg of 10% Palladium on charcoal (wet Degussa type) and thecontents of the flask was deoxygenated by vacuum and hydrogen purge. Itwas then pressurized to 60 Psi with hydrogen and stirred for 5 h at roomtemperature. The flask was then thoroughly flushed with argon andfiltered the solids through a short pad of celite. Evaporation andrecrystallization of the crude product afforded 900 mg (88%) of theCompound 2 in pure form as an off-white solid. ESMS calculated forC₂₃H₂₈N₄O₃: 408.22; Found: 409.1 (M⁺).

Synthesis: To a stirred solution of 0.1 g (0.245 mmols) of Compound 2 in5 mL of anhydrous N,N-Dimethylformamide was added in portion 0.13 g(0.245 mmols) of Compound 3(4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl(4-nitrophenyl) carbonate) and the mixture was stirred at roomtemperature for 2 h. After confirming the completion of the reaction byLC-MS, 30 mL of water was added to the flask and stirred for 5 mins. Theresultant precipitate was filtered, thoroughly washed with water (10mL×3) and dried. The solids were dissolved in 25 mL of95:5-dichloromethane:methanol and dried over anhydrous Na₂SO₄.Evaporation followed by column chromatography afforded the conjugate 1which was further purified by crystallization in methanol to removeminor impurities (mostly SN-38) and the procedure afforded 130 mg (65%)of the pure Conjugate 1. ¹H NMR (400 MHz, DMSO-d₆), δ (ppm): 11.93 (bs,1H), 9.57 (bs, 1H), 9.45 (bs, 1H), 8.18 (d, J=8 Hz, 1H), 7.98 (s, 1H),7.66 (dd, J=4.0, 8.0 Hz, 1H). 7.34 (s, 1H), 7.24 (d, J=8 Hz, 2H), 7.13(d, J=8 Hz, 2H), 6.77 (s, 1H), 6.54 (bs, 1H), 6.28 (s, 1H), 5.44 (s,2H), 5.34 (s, 2H), 3.21-3.18 (m, 2H), 3.10-2.96 (m, 3H), 2.59 (d, J=8Hz, 2H), 1.91-1.76 (m, 3H), 1.67 (bs, 2H), 1.30 (t, J=8 Hz, 3H), 0.95(d, J=8 Hz, 6H), 0.89 (d, J=8 Hz, 3H). ESMS calculated for C₄₆H₄₆N₆O₉:826.33; Found: 827.3 (M⁺).

Additional SDC-TRAPs made according to the general scheme noted aboveinclude the following:

Compound SDC-TRAP-0008:

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)carbamate

ESMS calculated for C₄₄H₄₁N₇O₉: 811.30; Found: 812.3 (M⁺).

SDC-TRAP-0015

N1-(2-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethoxy)ethyl)-N5-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)glutaramide

ESMS calculated for C₄₁H₄₄N₈O₉: 792.32; Found: 793.3 (M⁺).

SDC-TRAP-0016

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl(2-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethoxy)ethyl)carbamate

ESMS calculated for C₄₆H₄₅N₇O₁₀: 855.32; Found: 856.3 (M⁺).

SDC-TRAP-0017

3-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)propanamide

ESMS calculated for C₃₅H₃₃N₇O₇: 663.24; Found: 664.3 (M⁺).

SDC-TRAP-0018:

N1-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)-N5-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)-N1-methylglutaramide

ESMS calculated for C₄₀H₄₂N₈O₈: 762.31; Found: 763.3 (M⁺).

SDC-TRAP-0019:

4-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl)-N-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)-N-methylbenzamide

¹H NMR (300 MHz, DMSO-d6), d (ppm): 11.86 (s, 1H); 10.61 (s, 1H); 10.14(s, 1H); 9.51 (s, 1H); 9.47 (s, 1H); 7.59-7.45 (m, 2H); 7.28-6.96 (m,5H); 6.72 (m, 2H); 6.47 (s, 1H); 6.32 (s, 1H); 6.24 (s, 1H); 6.00 (bs,2H); 4.46-4.28 (m, 2H); 3.75-3.49 (m, 2H); 2.96-2.80 (m, 5H); 2.61 (s,3H); 0.81 (d, J=6.9 Hz, 6H). ESMS calculated for C₃₇H₃₇N₉O₅: 687.29;Found: 688.2 (M⁺).

SDC-TRAP-0020:

4-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl)-N-(2-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethoxy)ethyl)benzamide

ESMS calculated for C₃₈H₃₉N₉O₆: 717.3; Found: 718.3 (M⁺).

SDC-TRAP-0021:

2-(3-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)-3-methylureido)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)acetamide

ESMS calculated for C₃₈H₃₉N₉O₈: 749.29; Found: 750.3 (M⁺).

SDC-TRAP-0022:

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)(methyl)carbamate

ESMS calculated for C₄₅H₄₃N₇O₉: 825.31; Found: 826.3 (M⁺).

SDC-TRAP-0010:

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-N,1-dimethyl-1H-indole-2-carboxamido)ethyl)(methyl)carbamate

ESMS calculated for C₄₈H₄₈N₈O₁₀: 896.35; Found: 897.4 (M⁺).

SDC-TRAP-0023:

2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-N-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)acetamide

ESMS calculated for C₄₅H₄₃N₇O₉: 825.31; Found: 826.3 (M⁺).

SDC-TRAP-0027:

2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-N-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)-N-methylacetamide

ESMS calculated for C₄₆H₄₅N₇O₉: 839.33; Found: 840.4 (M⁺).

SDC-TRAP-0028:

2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-N-(2-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethoxy)ethyl)-N-methylacetamide

ESMS calculated for C₄₈H₄₉N₇O₁₀: 883.35; Found: 884.4 (M⁺).

SDC-TRAP-0029:

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl(2-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethoxy)ethyl)(methyl)carbamate

ESMS calculated for C₄₇H₄₇N₇O₁₀: 869.34; Found: 870.4 (M⁺).

SDC-TRAP-0031:

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperidine-1-carboxylate

¹H NMR (400 MHz, DMSO-d₆), d (ppm): 11.93 (bs, 1H), 9.57 (bs, 1H), 9.45(bs, 1H), 8.18 (d, J=8 Hz, 1H), 7.98 (s, 1H), 7.66 (dd, J=4.0, 8.0 Hz,1H). 7.34 (s, 1H), 7.24 (d, J=8 Hz, 2H), 7.13 (d, J=8 Hz, 2H), 6.77 (s,1H), 6.54 (bs, 1H), 6.28 (s, 1H), 5.44 (s, 2H), 5.34 (s, 2H), 3.21-3.18(m, 2H), 3.10-2.96 (m, 3H), 2.59 (d, J=8 Hz, 2H), 1.91-1.76 (m, 3H),1.67 (bs, 2H), 1.30 (t, J=8 Hz, 3H), 0.95 (d, J=8 Hz, 6H), 0.89 (d, J=8Hz, 3H). ESMS calculated for C₄₆H₄₆N₆O₉: 826.33; Found: 827.3 (M⁺).

SDC-TRAP-0024

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1-methyl-1H-indole-2-carboxamido)butanoate

¹H NMR (400 MHz, CH₃OD) δ 7.88 (d, J=8.0 Hz, 1H), 7.44 (s, 1H),7.35-7.27 (m, 4H), 7.16-7.14 (m, 1H), 6.73 (s, 1H), 6.67 (s, 1H), 6.26(s, 1H), 5.62 (d, J=16 Hz, 1H), 5.44 (d, J=16 Hz, 1H), 5.05 (d, J=16 Hz,1H), 3.58 (s, 3H), 3.48-3.33 (m, 3H), 3.09-3.04 (m, 1H), 2.96-2.86 (m,2H), 2.75-2.71 (m, 2H), 2.25-2.13 (m, 2H), 2.05-1.94 (m, 2H), 1.29 (t,J=8.0 Hz, 3H), 1.01 (t, J=8.0 Hz, 3H), 0.78-0.72 (m, 6H); ESMScalculated for C₄₇H₄₅N₇O₁₀: 867.3; found: 868.3 (M+H).

SDC-TRAP-0025:

2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl(5-fluoro-2-oxo-1,2-dihydropyrimidin-4-yl) carbamate

ESMS calculated C₂₆H₂₄FN₇O₆: 549.18; found: 550.1 (M+H).

SDC-TRAP-0033:

N1-(2-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethoxy)ethyl)-N₄-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)-N1-methylsuccinamide

ESMS calculated for C₄₁H₄₄N₈O₉: 792.32; found: 793.3 (M+H).

SDC-TRAP-0037:

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl(2-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethoxy)ethyl)(methyl)carbamate

ESMS calculated for C₄₇H₄₇N₇O₁₀: 869.34; found: 870.3 (M+H).

SDC-TRAP-0038:

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)(methyl)carbamate

ESMS calculated for C₄₅H₄₃N₇O₉: 825.31; found: 826.3 (M+H).

SDC-TRAP-0039:

4-(5-(bis(2-chloroethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)-N-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)-N-methylbutanamide

ESMS calculated for C₃₈H₄₄Cl₂N₈O₄: 746.29; found: 747.3 (M+H).

SDC-TRAP-0040:

4-(5-(bis(2-chloroethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)-N-(2-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethoxy)ethyl)-N-methylbutanamide

ESMS calculated for C₄₀H₄₈Cl₂N₈O₅: 790.31; found: 791.3 (M+H).

SDC-TRAP-0041:

5-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)-5-oxopentanamide

ESMS calculated for C₄₀H₄₄N₈O₈: 764.33; found: 765.3 (M+H).

SDC-TRAP-0042:

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperidin-1-yl)-4-oxobutanoate

ESMS calculated for C₄₉H₅₀N₆O₁₀: 882.36; found: 883.3 (M+H).

SDC-TRAP-0043:

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)-4-oxobutanoate

ESMS calculated for C₄₈H₄₉N₇O₁₀: 883.35; found: 884.3 (M+H).

SDC-TRAP-0044:

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl(4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)butyl)(methyl)carbamate

ESMS calculated for C₅₀H₅₆N₈O₉: 912.42; found: 913.4 (M+H).

SDC-TRAP-0045:

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenyl)piperazine-1-carboxylate

ESMS calculated for C₄₄H₄₃N₇O₉: 813.31; found: 814.3 (M+H).

SDC-TRAP-0046:

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazine-1-carboxylate

ESMS calculated for C₄₅H₄₅N₇O₉: 827.33; found: 828.3 (M+H).

SDC-TRAP-0047:

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazine-1-carboxylate

ESMS calculated for C₄₅H₄₅N₇O₉: 827.33; found: 828.3 (M+H).

SDC-TRAP-0048:

N-(2-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethoxy)ethyl)-3-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)propanamide

ESMS calculated for C₃₀H₃₂FN₇O₇: 621.23; found: 622.2 (M+H).

SDC-TRAP-0049:

1-(3-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)-3-oxopropyl)-5-fluoropyrimidine-2,4(1H,3H)-dione

ESMS calculated for C₂₉H₃₂FN₇O₆: 593.24; found: 594.2 (M+H).

SDC-TRAP-0050:

N-(2-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethoxy)ethyl)-3-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N-methylpropanamide

ESMS calculated for C₃₁H₃₄FN₇O₇: 635.64; found: 636.6 (M+H).

SDC-TRAP-0051:

N-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)-3-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N-methylpropanamide

ESMS calculated for C₂₉H₃₀FN₇O₆: 591.22; found: 592.2 (M+H).

Example 2

The ability of Hsp90-targeting moieties to penetrate solid tumors andexhibit rapid clearance from normal tissues for reduced toxicity isillustrated in the following tissue distribution study with a compound,ganetespib, which may be used as an Hsp90 binding moiety.

Tissue Distribution of Ganetespib in Female CD-1 Nu/Nu Mice Bearing RERFHuman NSCLC Xenografts

Objectives:

To confirm the distribution of ganetespib in blood, livers, kidneys,brains, hearts, lungs and tumors after IV administration of ganetespibto female CD-1 nu/nu mice bearing RERF human NSCLC xenografts, and toexamine metabolic profiles of ganetespib in plasma, red blood cells, andabove tissues.

Study Outline:

Test Articles: ganetespib

Animals: female CD-1 nu/nu mice bearing RERF human NSCLC xenografts(N=3/group)

Route: IV

Dosage: 50 mg/kg

Dose level: 10 mL/kg

Formulation: 10% DMSO, 18% Cremophor RH40, 3.6% dextrose solution (DRD)

Bleeding time points: 5 min, 6, 24 hr

Collected tissues: blood (plasma and red blood cells (RBC)), liver,kidneys, brain, heart, lung, tumor

Method Sample Preparation

Plasma and RBC

Protein precipitation: 50 μL of 10 times diluted plasma or RBC+150 μLACN (10 mM NH₄OAc), vortexed and centrifuged at 10000 rpm for 8 min; 150μL supernatant+150 μL water (10 mM NH₄OAc)

Other Tissues

Protein precipitation: 100 μL homogenized tissue (1:3 tissue: PBSbuffer)+100 μL ACN (10 mM NH₄OAc), vortexed and centrifuged at 10000 rpmfor 8 min

Bioanalysis

HPLC (ChemStation)

Column: Agilent Zorbax Eclipse XDB-C18, 4.6×150 mm, 5 μm

Mobile phase: A: water containing 10 mM NH₄OAc; B: 95% ACN containing 10mM NH₄OAc

Gradient: 95/5 A/B to 5/95 A/B in 10 min, total run time 15 min

Flow rate: 1 mL/min

Column temp.: 40° C.

Wavelength: 254 nm

Injection volume: 100 μL

Calibration curve range:

Plasma: 1-50 μM (linear regression; R²=0.9901); LLOQ=1 μM

RBC: 1-50 μM (linear regression; R²=0.9987); LLOQ=1 μM

Kidney: 1-100 μM (linear regression; R²=1.0000); LLOQ=1 μM

Lung: 1-100 μM (linear regression; R²=1.0000); LLOQ=1 μM

Heart: 1-100 μM (linear regression; R²=0.9998); LLOQ=1 μM

Liver: 1-100 μM (linear regression; R²=1.0000); LLOQ=1 μM

Tumor: 0.1-10 μM (linear regression; R²=1.0000); LLOQ=0.1 μM

LC-MS/MS (Q-Trap4000)

Polarity: positive (ESI)

Column: Phenomenex Synergi, 2.1×50 mm, 4 μm

Mobile phase: A: water containing 0.1% HCOOH; B: ACN containing 0.1%HCOOH

Gradient: 60/40 A/B to 5/95 A/B in 0.5 min, total run time 4 min

Flow rate: 0.5 mL/min

Column temp.: room temperature

Injection volume: 20 μL

Calibration curve range:

Plasma: 2.5-500 nM (linear regression; R²=0.9994); LLOQ=2.5 nM

RBC: 2.5-500 nM (linear regression; R²=0.9998); LLOQ=2.5 nM

Kidney: 2.5-500 nM (linear regression; R²=0.9993); LLOQ=2.5 nM

Lung: 2.5-500 nM (linear regression; R²=0.9993); LLOQ=2.5 nM

Heart: 2.5-500 nM (linear regression; R²=0.9997); LLOQ=2.5 nM

Liver: 2.5-500 nM (linear regression; R²=1.0000); LLOQ=2.5 nM

-   -   0.5-5 μM (linear regression; R²=0.9970); LLOQ=0.5 μM

Brain: 2.5-500 nM (linear regression; R²=0.9998); LLOQ=2.5 nM

-   -   0.5-5 μM (linear regression; R²=0.9992); LLOQ=0.5 μM

Results Formulations

The dosing solution was confirmed to have 98.1% accuracy by HPLC.

Tissue Distribution

The concentrations of ganetespib in plasma, RBC and the tissues aresummarized in Table 1 and FIG. 1 at each time point.

The mean plasma concentration of ganetespib at 5 min after IV injectionwas 160 μM, highest among all the tissues studied. Thereafter, theplasma ganetespib concentration declined quickly and at 6 hr, it was0.12 μM. At 24 hr, it was below the lower limit of quantitation (LLOQ,<2.5 nM).

After IV injection, ganetespib was widely distributed to the normaltissues analyzed. At 5 min, the highest concentration of ganetespibamong the tissues was observed in kidney (57.8 μM), followed by liver(46.3 μM) and heart (36.2 μM). In brain, 0.53 μM of ganetespib wasdetected at 5 min, which was the lowest among the tissues. In all thenormal tissues, the concentrations of ganetespib decreased quickly.

Although the concentration of ganetespib in tumor at 5 min (2.35 M) waslower than that in plasma and most of the other tissues studied, itremained relatively constant up to 24 hr (0.85 μM at 24 hr). However,the in vitro IC₅₀ values of ganetespib are small, and the tumorconcentration of ganetespib at 24 hr was significantly higher than IC₅₀of in vitro HER2 assays (˜30 nM). Thus, the prolonged efficacy isexpected even after ganetespib was cleared from the blood stream.

The mean concentration of ganetespib in plasma was about 10 times higherthan that in RBC at 5 min time point, indicating that ganetespib tendsto stay in plasma rather than in RBCs. See FIG. 3.

CONCLUSION

Ganetespib appeared to persist longer in tumor than in plasma or anyother tissues studied. The results from this study suggest thatganetespib also has a higher binding affinity to Hsp90 from tumor cellsthan Hsp90 from normal cells, and that it is possible for ganetespib tomodulate relative protein concentrations of Hsp90 and its clientproteins selectively in tumors. The plasma concentrations of ganetespibdid not correlate to the concentrations in tumor.

TABLE 1 Concentrations of ganetespib in tissues: Test Articlesganetespib Structure

Species CD-1-nu/nu female mice Tumor RERF human NSCLC Route IV Dosage 50mg/kg Formulation DRD plasma RBC tumor liver kidneys brain heart lungTime (μg/mL) (μg/mL) (μg/g) (μg/g) (μg/g) (μg/g) (μg/g) (μg/g)  5 min58.4 6.00 0.86 16.9 21.1 0.19 13.2 9.24  6 hr 0.04 No data 0.29 0.140.06 0.07 0.05 0.05 24 hr <LLOQ 0.003 0.31 0.005 0.01 0.04 0.00 0.00plasma RBC tumor liver kidneys brain heart lung Time (μM) (μM) (μM) (μM)(μM) (μM) (μM) (μM)  5 min 160 16.5 2.35 46.3 57.8 0.53 36.2 25.4  6 hr0.12 N/A 0.80 0.39 0.15 0.18 0.13 0.14 24 hr <LLOQ 0.007 0.85 0.01 0.020.12 0.00 0.005

SUMMARY

Ganetespib was widely distributed to various tissues. The compound wasaccumulated in tumor relative to the plasma and other tissues,indicating the higher binding affinity of this compound to Hsp90 intumor than Hsp90 in other tissues. The metabolite M2, which waspreviously thought to be human-specific, was also detected in mouseliver, kidney, heart and lung, but not in plasma. M2 does not seem to beexcreted into blood stream in mice and possibly in other species aswell.

Example 3

This example illustrates how a HER2 degradation assay may be used as atest to determine and select Hsp90-targeting moieties suitable for usein SDC-TRAPs of the invention, and further illustrates the ability ofSDC-TRAPs to target cells preferentially expressing Hsp90. Such a testmay further be used to determine the Hsp90 binding ability of SDC-TRAPsof the invention, as well as through competitive binding assays andcell-based Hsp90 client protein degradation assays known in the art.

Degradation of HER2 in Cells after Treatment with an SDC-TRAP of theInvention

Method 1:

BT-474 cells are treated with 0.5 μM, 2 μM, or 5 μM of 17-AAG (apositive control) or 0.5 μM, 2 μM, or 5 μM of an Hsp90-targeting moietyor conjugate of the invention overnight in DMEM medium. After treatment,each cytoplasmic sample is prepared from 1×10⁶ cells by incubation ofcell lysis buffer (#9803, Cell Signaling Technology) on ice for 10minutes. The resulting supernatant used as the cytosol fractions isdissolved with sample buffer for SDS-PAGE and run on a SDS-PAGE gel,blotted onto a nitrocellulose membrane by using semi-dry transfer.Non-specific binding to nitrocellulose is blocked with 5% skim milk inTBS with 0.5% Tween at room temperature for 1 hour, then probed withanti-HER2/ErB2 mAb (rabbit IgG, #2242, Cell Signaling) and anti-Tubulin(T9026, Sigma) as housekeeping control protein. HRP-conjugated goatanti-rabbit IgG (H+L) and HRP-conjugated horse anti-mouse IgG (H+L) areused as secondary Ab (#7074, #7076, Cell Signaling) and LumiGLO reagent,20× Peroxide (#7003, Cell Signaling) is used for visualization. TheHsp90 client protein HER2 is degraded when cells are treated withHsp90-targeting moieties or SDC-TRAPs of the invention. 0.5 μM of17-AAG, a known Hsp90 inhibitor used as a positive control, causespartial degradation of HER2.

Method 2:

BT-474 cells are plated in the interior 60 wells of a 96 well blackclear bottom plate (20,000 cells/well) in DMEM medium, with DMEM mediain the surrounding 36 wells, and incubated at 37° C. with 5% CO₂overnight. On the second day, concentration response curve source platesare produced (10 point, 3-fold dilution of compounds in DMSO) followedby a 1:30 dilution in an intermediate dilution plate containing DMEM.Compound is transferred from the intermediate plate to the cell plate ata dilution of 1:10. The cells are then incubated at 37° C. with 5% CO₂for 24 hours.

Cells are then fixed in 4% phosphate-buffered paraformaldehyde for 30minutes at room temperature and then permeabilized by washing five timeswith 0.1% Triton X-100 in PBS for 5 minutes at room temperature on ashaker. Cells are blocked with Odyssey Blocking Buffer (LI-COR,#927-40000) on a shaker at room temperature for 1.5 hours, followed byincubation with HER2 antibody (CST, #2165) diluted 1:400 in blockingbuffer overnight on a shaker at 4° C. Cells are washed five times with0.1% Tween-20 in PBS for 5 minutes at room temperature on a shaker andincubated with fluorescently-labeled secondary antibody (LI-COR,#926-32211) diluted 1:1000 in blocking buffer, and DRAQ5 nuclear stain(Biostatus Limited, #DRAQ5) diluted 1:10,000, at room temperature on ashaker for 1 hour. Cells are washed 5 times with 0.1% Tween-20 in PBSfor 5 minutes at room temperature on a shaker and imaged on a LI-COROdyssey imaging station. The raw data is normalized to DRAQ5 and theHER2 EC₅₀ is calculated using XLfit™.

The above procedures were utilized to generate the following HER2degradation data, which show the ability of these exemplary SDC-TRAPs totarget cells preferentially expressing Hsp90:

HER2 (IC₅₀, nM) SDC-TRAP 2347 SDC-TRAP-0015 >5000 SDC-TRAP-0017 >5000SDC-TRAP-0018 4419 SDC-TRAP-0019 >5000 SDC-TRAP-0020 >5000SDC-TRAP-0021 >5000 SDC-TRAP-0022 >5000 SDC-TRAP-0010 4300SDC-TRAP-0023 >5000 SDC-TRAP-0027 >5000 SDC-TRAP-0028 1603 SDC-TRAP-00292916 SDC-TRAP-0031 >5000 SDC-TRAP-0024 395 SDC-TRAP-0025 >5000SDC-TRAP-0033 2112 SDC-TRAP-0037 >5000 SDC-TRAP-0038 2935 SDC-TRAP-00394741 SDC-TRAP-0040 >5000 SDC-TRAP-0041 1057 SDC-TRAP-0042 2135SDC-TRAP-0043 602 SDC-TRAP-0044 464 SDC-TRAP-0045 246 SDC-TRAP-0046 875SDC-TRAP-0047

Example 4

This example illustrates a method of assessing the cytotoxicity ofSDC-TRAPs of the invention.

Cell Lines.

Human H3122 NSCLC cells were obtained and grown in RPMI in the presenceof fetal bovine serum (10%), 2 mM L-glutamine and antibiotics (100 IU/mlpenicillin and 100 μg/ml streptomycin, Sigma Aldrich.) Cells weremaintained at 37° C., 5% CO₂ atmosphere.

Cell Viability Assays.

Cell viability was measured using the CELLTITER GLO assay (Promega). Inbrief, cells were plated in 96-well plates in triplicate at optimalseeding density (determined empirically) and incubated at 37° C., 5% CO₂atmosphere for 24 hr prior to the addition of drug or vehicle (0.3%DMSO) to the culture medium. At the end of the assay, CELLTITER GLO wasadded to the wells per manufacturer's recommendation, shaken for twominutes and incubated for 10 minutes at room temperature. Luminescence(0.1 sec) was measured with a Victor II microplate reader (Perkin Elmer)and the resulting data were used to calculate cell viability, normalizedto vehicle control.

Cells as described above were treated with exemplary SDC-TRAPs and theirviability determined as above as well. The following table illustratesthe results.

IC₅₀ (H3122) SDC-TRAP Number (nM) SDC-TRAP-0010 234 SDC-TRAP-0015 1273SDC-TRAP-0017 >3000 SDC-TRAP-0018 620 SDC-TRAP-0019 393 SDC-TRAP-00201737 SDC-TRAP-0021 717 SDC-TRAP-0022 492 SDC-TRAP-0023 137 SDC-TRAP-002499 SDC-TRAP-0027 1354 SDC-TRAP-0028 909 SDC-TRAP-0029 125

Example 5

This example illustrates a method for assessing the stability ofSDC-TRAP of the invention in human and mouse plasma.

SDC-TRAP-0022 and SDC-TRAP-0028 were incubated in human and mouse plasmafor 2 h at 37° C. and assayed for integrity at 0.25, 0.5, 1 and 2 h. Thevalues reported below are the remaining of the parent compound at theend of the 2 h incubation period.

% Remaining 2 h (37° C.) Conjugate ID Concentration HU MO SDC-TRAP-0022 1 μM 29% 32% 10 μM 30% 31% SDC-TRAP-0028  1 μM 51% 53% 10 μM 65% 47%

Example 6

A detailed schematic for the synthesis of SDC-TRAP-0063

A detailed scheme of the synthesis of SDC-TRAP-0063 is provided. Theperson of ordinary skill in the art would be able, without undueexperimentation, to adapt this synthetic scheme for making othertargeting molecule conjugates within the scope of the invention.

As explained hereinabove, SDC-TRAP-0063 is essentially a conjugate ofthe binding moiety ganetespib and the effector moiety irinotecan.SDC-TRAP-0063 is:4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)piperidine-1-carboxylate.

SDC-TRAP-0063 was synthesized according to the following scheme:

Synthesis of each of the above intermediates (INT) is detailed asfollows.

Preparation of tert-butyl 4-(2-hydroxyethyl)piperidine-1-carboxylate(INT-1)

To a stirred solution of 2-(piperidin-4-yl)ethanol (30 g, 0.2322 mmol)in 1,2-dichloromethane (200 ml) was added in portions di-tert-butyldicarbonate (53 g, 0.24 mmol). The resultant mixture was stirred at roomtemperature overnight. After confirming reaction completion bythin-layer chromatography, the reaction mixture was washed with waterand concentrated to yield compound INT-1 (52 g).

Preparation of tert-butyl4-(2-((methylsulfonyl)oxy)ethyl)piperidine-1-carboxylate (INT-2)

To a stirred solution of INT-1 (52 g, 0.23 mmol), 4-dimethylaminopyridine (4.2 g, 3.41 mmol) and triethylamine (92 g, 908 mmol) in1,2-dichloroethane was added to methanesulfonyl chloride drop wise at 0°C., and the mixture was stirred at room temperature overnight. Afterconfirming reaction completion by thin-layer chromatography, the mixturewas washed with water and concentrated to yield compound INT-2 (67 g).

Preparation of tert-butyl4-(2-(5-nitro-1H-indol-1-yl)ethyl)piperidine-1-carboxylate (INT-3)

To a stirred solution of 5-nitro-1H-indole (SM-2, above, 30 g, 185 mmol)in N,N-dimethylformamide (200 ml), sodium hydride (13 g, 325.5 mmol) wasadded in portions at 0° C. and the mixture was stirred at roomtemperature for 30 min. INT-2 (67 g, 217 mmol) was added at 0° C. andthe resultant mixture was stirred at room temperature overnight. Themixture was carefully poured into ice water while a yellow precipitatewas observed. The mixture was extracted with ethyl acetate followeddrying and concentration to afford the crude product, which was thenpurified by silica gel chromatography to yield INT-3 as a yellow solid(80 g).

Preparation of compound tert-butyl 4-(2-(5-amino-1H-indol-1-yl)ethyl)piperidine-1-carboxylate (INT-4)

To a solution of INT-3 (80 g, 215 mmol) in a mixture of ethanol (200 ml)and tetrahydrofuran (350 ml) was added Raney nickel (10 g). Theresultant mixture was stirred at room temperature overnight underhydrogen atmosphere. The contents then were filtered to remove thesolids and concentrated to yield INT-4 (70 g).

Preparation of compound tert-butyl4-(2-(5-(2,4-dihydroxy-5-isopropylphenylthioamido)-1H-indol-1-yl)ethyl)piperidine-1-carboxylate(INT-5)

A mixture of 2,4-dihydroxy-5-isopropylbenzodithioic acid (SM-3, 46.5 g,204 mmol), sodium 2-chloroacetate (38 g, 326.4 mmol) and sodiumbicarbonate (52.0 g, 612 mmol) in N,N-dimethylformamide (350 ml) wasdegassed using nitrogen gas to remove oxygen. The reaction mixture thenwas stirred at 25° C. for 3 hours. The second reactant, INT-4 (70.0 g,204 mmol) in N,N-dimethylformamide (150 ml) was added slowly to thereaction mixture through a syringe. The reaction mixture was stirred at80° C. for 3 hours. After reaction completion, the reaction mixture wasextracted with ethyl acetate, washed with water, then brine, and dried.Concentration by flash chromatography yielded INT-5 (58 g).

Preparation of tert-butyl4-(2-(5-(7-hydroxy-6-isopropyl-2-oxo-4-thioxo-2H-benzo[e][1,3]oxazin-3(4H)-yl)-1H-indol-1-yl)ethyl)piperidine-1-carboxylate(INT-6)

To a stirred solution of compound INT-5 (27 g, 50.86 mmol) intetrahydrofuran (200 ml), carbonyldiimidazole (16.5 g, 101.7 mmol) wasadded in portions. The resulting mixture was stirred at room temperaturefor 3 hours under nitrogen atmosphere, then poured into water andextracted with ethyl acetate. The organic layer was dried over anhydrousNa₂SO₄ and concentrated to yield INT-6 (28 g).

Preparation of tert-butyl4-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)piperidine-1-carboxylate(INT-7)

To a stirred solution of compound INT-6 (28 g, 50.86 mmol) in anhydrousethanol (200 mL) was added hydrazine hydrate (5 ml, 102.2 mmol), and theresulting mixture was stirred overnight at room temperature under argonatmosphere. The reaction product was filtered over a short pad of silicagel, followed by concentration and thorough drying yielding INT-7 (16.4g.)

Preparation of4-(5-hydroxy-4-(1-(2-(piperidin-4-yl)ethyl)-1H-indol-5-yl)-4H-1,2,4-triazol-3-yl)-6-isopropylbenzene-1,3-diol(INT-8)

To a solution of compound INT-7 (8 g, 14.3 mmol) in methanol (40 mL) wasadded a solution of 1.0 M HCl in methanol (100 ml). The resultingmixture was stirred at room temperature overnight. The resultant solidswere concentrated, then washed with methanol to yield INT-8 as ahydrochloride salt (4.8 g.)

To a 0° C. stirred suspension of4-(5-hydroxy-4-(1-(2-(piperidin-4-yl)ethyl)-1H-indol-5-yl)-4H-1,2,4-triazol-3-yl)-6-isopropylbenzene-1,3-diolhydrochloride (INT-8, 3.0 mmol) and(S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl(4-nitrophenyl) carbonate (INT-9, 3.0 mmol) in dimethylformamide (40 mL)was added triethylamine (4.0 mmol) dropwise, and the mixture was stirredat 0° C. for 1 hour. 50 mL water then was poured into the mixture. Theyellow suspension was stirred at room temperature for 1 hour, thenfiltered. The filter cake was washed with water (10 mL×2) and purifiedby column chromatography to yield SDC-TRAP-0063 as a white solid (2.20g, 2.5 mmol).

¹H NMR (400 MHz, Chloroform-d) δ 8.21 (d, J=9.2 Hz, 1H), 7.84 (d, J=2.5Hz, 1H), 7.68 (s, 1H), 7.64-7.56 (m, 2H), 7.47 (d, J=8.7 Hz, 1H),7.24-7.12 (m, 2H), 6.55 (dd, J=3.2, 0.8 Hz, 1H), 6.37 (d, J=4.2 Hz, 2H),5.73 (d, J=16.3 Hz, 1H), 5.36-5.24 (m, 3H), 4.41 (d, J=13.5 Hz, 1H),4.29 (q, J=9.3, 7.5 Hz, 3H), 3.17 (q, J=7.7 Hz, 2H), 3.06 (t, J=12.7 Hz,1H), 2.96-2.77 (m, 2H), 2.42 (s, 2H), 1.90 (dq, J=14.2, 7.1 Hz, 6H),1.45-1.33 (m, 5H), 1.31-1.22 (m, 1H), 1.04 (t, J=7.3 Hz, 3H), 0.50 (d,J=6.8 Hz, 6H). ppm; ESMS calculated for C₄₉H₄₉N₇O₉: 879.4; found: 880.2(M+H⁺).

Example 7

The following example uses a number of assays to characterizeSDC-TRAP-0063 (described in Example 6.)

In vitro activity as determined by the HER2 degradation and Hsp90binding assay is set forth below. Protocols for the HER2 degradationassay and Hsp90 binding assay are provided in Examples 11 and 12,respectively.

IC₅₀ (HER2 degradation assay) EC₅₀ (Hsp90 binding assay) 793 nM 157 nM

In order to determine the stability of SDC-TRAP-0063 in plasma, thecompound was exposed to mouse plasma and the percent of the compoundremaining at 1 hour was determined. After 1 hour 11.1% of SDC-TRAP-0063remained. As shown below, SDC-TRAP-0063 breaks down into degradationproduct 1 (DP-1, an Hsp90 inhibitor fragment) and SN-38.

The degradation of SDC-TRAP-0063 was followed in mouse plasma. Therelease profile of fragment DP-1 and payload (SN-38) was determinedaccording to the protocols provided in Examples 16-18.

MOUSE PLASMA (MO) Peak Area Ratio Time (h) 0 0.25 0.5 1 SDC-TRAP-006317.9 15.6 7.77 1.98 DP-1 0.00133 0.00268 0.0190 0.113 SN-38 0.0616 1.374.13 4.46

In order to determine if SDC-TRAP-0063 is targeting tumor cellsselectively, the tissue distribution of SDC-TRAP-0063 and itsdegradation products DP-1 and SN-38 was monitored in mouse plasma, tumorand heart. Data from these experiments are presented in the table belowand in FIGS. 15A-C. The data demonstrate that SDC-TRAP-0063 selectivelytargets and accumulates in tumor cells, as does the degradation productsof SDC-TRAP-0063 including the chemotherapeutic SN-38.

Compound ID SDC-TRAP-0063 Lot 1 Dose 50 mg/10 mL/kg Species Female SCIDMouse (H1975) Route IV Formulation DRD Appearance N/A Accuracy N/AAnalyte Target SDC-TRAP-0063 DP-1 SN-38 Time (h) Plasma Conc. (μM) 0.083526 0.0662 20.4 6 1.69 0.0397 0.0509 24 0.00675 0.0175 0.0240 48 BQL0.00793 0.00524 Time (h) Tumor Conc. (nmol/g of tissue) 0.083 6.430.00758 1.47 6 1.61 0.111 0.730 24 0.203 0.404 0.618 48 0.0188 1.060.296 Time (h) Heart Conc. (nmol/g of tissue) 0.083 79.1 0.0271 0.927 60.536 0.207 BQL 24 BQL 0.0855 BQL 48 BQL 0.0238 BQL

Mouse Xenograft Efficacy Data in an HCT-116 Colon Cancer Model

A xenograft tumor model was used to evaluate the anti-tumor efficacy ofSDC-TRAP-0063. The tumor model was established by transplanting HCT-116tumor cells into mice and testing the effect of SDC-TRAP-0063 on tumorvolume and change in tumor volume.

HCT 116 human colorectal adenocarcinoma tumor cells were purchased fromATCC. The cells were maintained in vitro as a monolayer culture inMcCoy's 5a Medium. Fetal bovine serum was then be added to the medium.The final concentration of fetal bovine serum was 10%. Cells werecultured at 37° C. and 5% CO₂. The tumor cells were routinelysub-cultured twice weekly by trypsin-EDTA treatment. Cells in anexponential growth phase were harvested and counted for tumorinoculation.

100 18-22 g, 5-7 week old, female BALB/cA nude mice were inoculated withthe HCT 116 cells (2.0×10⁶, 1:1 with Matrigel) subcutaneously on theback of each animal (0.1 mL/mouse). When the average tumor volumereached about 150-250 mm³, 60 of the inoculated mice was selected basedon tumor growth and randomly grouped into 6 treatment groups (10 miceper group) according to the following table. Mice that were not put ontreatment were euthanized. Animals were sourced through ShanghaiSINO-British SIPPR/BK Lab Animal Ltd, Shanghai, China. Mice were treatedas set fotth in the table beow:

Treatment Groups:

Dosage Dosage Route Animal Dosage Conc. Vol. of Dosing Groups NumberTreatment (mg/kg) (mg/mL) (mL/kg) Adm. Schedule 1 10 Vehicle NA NA 10 IVQ7D × 3 2 10 SDC-TRAP- 200 20 10 IV Q7D × 3 0063 3 10 SDC-TRAP- 100 1010 IV Q7D × 3 0063 4 10 SDC-TRAP- 94 9.4 10 IV Q7D × 3 0046 5 10irinotecan 67 6.7 10 IV Q7D × 3 6 10 irinotecan 67 6.7 10 IV Q7D × 3 7SYN-01 100 10 10 IV Q7D × 3

Dose Preparation & Treatment Schedule:

The dosing solutions of SDC-TRAP-0063, SDC-TRAP-0046, SYN-01(ganetespib)and irinotecan were prepared according to the DRD formulation protocol(10% dimethyl sulfoxide (DMSO), 18% Cremophore RH40, 3.6% dextrose,68.4% sterile water and the clearly dissolved drug was added at desiredconcentration in DMSO). The administrations were made with 27-gauge IVneedle.

Evaluation of Anti-Tumor Activity:

During the treatment period, the implanted tumors were measured bycaliper twice per week. The tumors were measured for the maximum width(X) and length (Y) and the tumor volumes (V) were calculated using theformula: V=(X²Y)/2. The differences in the tumor volume between thecontrol and treatment groups were analyzed for significance using theunpaired two-tailed Student's t-test. P<0.05 was considered to bestatistically significant. The animal body weights were also weighed andrecorded twice per week. The changes in tumor volume in the daysfollowing compound treatment are shown in FIG. 4. The changes in animalbody weight in the days following compound treatment are shown in FIG.5.

Mouse Xenograft Efficacy Data in an MCF-7 Breast Cancer Model:

A xenograft tumor model to evaluate the anti-tumor efficacy ofSDC-TRAP-0063 was established by transplanting MCF-7 breast cancer cellsinto mice and testing the effect of SDC-TRAP-0063 on tumor volume andchange in tumor volume.

MCF-7 breast cancer cells were purchased from ATCC. The cells weremaintained in vitro as a monolayer culture in McCoy's 5a Medium. Fetalbovine serum was then added to the medium. The final concentration offetal bovine serum was 10%. Cells were cultured at 37° C. and 5% CO₂.The tumor cells were routinely sub-cultured twice weekly by trypsin-EDTAtreatment. Cells in an exponential growth phase were harvested andcounted for tumor inoculation.

75 24-30 g, 10-13 week old, female CD-1 nude mice were inoculated withthe MCF-7 cells (5.0×10⁶/mouse) orthotopically in mammary fat pad (0.1mL/mouse). 60 days estrogen pellets was implanted the day prior to cellimplantations. When the average tumor volume reached about 100-225 mm³,40 of the inoculated mice were selected based on tumor growth andrandomly grouped into 5 treatment groups (8 mice per group) according tothe following table. Mice that were not put on treatment wereeuthanized. Animals were sourced through CRL (Wilmington, Mass.).Animals were treated as set forth in the table below.

Dosage Dosage Animal Dosage Conc. Vol. Roe Dosing Groups NumberTreatment (mg/kg) (mg/mL) (mL/kg) Adm. Schedule 1 8 Vehicle NA NA 10 IVQ7D × 3 2 8 SDC-TRAP-0063 150 15 10 IV Q7D × 3 3 8 SDC-TRAP-0063 100 1010 IV Q7D × 3 5 8 Irinotecan 67 6.7 10 IV Q7D × 3 6 8 Irinotecan 67 6.710 IV Q7D × 3 ganetespib 42 10 10 IV Q7D × 3

Dose Preparation & Treatment Schedule:

The dosing solutions of SDC-TRAP-0063, ganetespib and irinotecan wereprepared in a standard DRD formulation (10% DMSO, 18% Cremophor RH40,3.6% dextrose, 68.4% sterile water, while clearly dissolved drugsubstances were added in DMSO.) The administrations were made with a27-gauge IV needle. In the combo group, irinotecan was dosed 2 hoursafter ganetespib.

Evaluation of Anti-Tumor Activity:

During the treatment period, the implanted tumors were measured bycaliper twice per week. The tumors were measured for the maximum width(X) and length (Y) and height (Z), the tumor volumes (V) were calculatedusing the formula: V=0.5236*X*Y*Z. The differences in the tumor volumebetween the control and treatment groups were analyzed for significanceusing % T/C value. Animal body weights were also weighed and recorded 5×per week. The changes in tumor volume in the days following compoundtreatment are shown in FIG. 6. The changes in animal body weight in thedays following compound treatment are shown in FIG. 7.

Preliminary Toxicological Evaluation Data (TK Analysis, BiomarkerAnalysis for Myelosuppression at Various Dose Levels in Rats):

The datat presented in FIG. 8 indicates that a higher dose (150mg/kg/1×wk) of conjugate SDC-TRAP-0063 appears to prolong thesuppression of increase in tumor volume compared to a lower dose (100mg/kg/1×wk). Either dose of SDC-TRAP-0063 has greater tumor growthsuppression than effector moiety irinotecan alone, or unconjugatedbinding moiety ganetespib and effector moiety irinotecan administeredtogether.

Example 8 Synthesis and Testing of Lenalidomide Conjugate SDC-TRAP-0178

Synthesis and testing of SDC-TRAP-0178, which is a conjugate of HSP90inhibitor fragment 3 and lenalidomide, is exemplified below.

Synthesis and Structure of Lenalidomide Conjugate SDC-TRAP-0178:

STEP-1: To a stirred suspension of lenalidomide 1 (520 mg, 2 mmol) indry THF (70 mL) was added 4-nitrophenylchloroformate (605 mg, 3 mmol).The reaction mixture was refluxed for 2 h, concentrated to approximately40 mL, and triturated with ethyl acetate to yield a white precipitate.The solid was collected by filtration and washed with ethyl acetate togive carbamate 2 (650 mg, 77%).

STEP-2: Diisopropylethylamine (33 mg, 0.25 mmol) was added to a stirredsolution of Hsp90 inhibitor fragment 3 (120 mg, 0.2 mmol) and theactivated lenalidomide 2 (86 mg, 0.2 mmol) in anhydrous DMF (5 mL). Thereaction mixture was stirred at room temperature for 18 h; then dilutedwith water (5 mL) and extracted with ethyl acetate (100 mL). The organicphase was dried (sodium sulfate), filtered and evaporated, followed byflash chromatography (hexane-ethyl acetate 1:1 and ethylacetate-methanol 98:2) to give SDC-TRAP-0178 (95 mg, 53%) as a whitesolid.

¹H NMR (400 MHz, DMSO-d₆) δ 11.02 (s, 1H), 10.22 (s, 1H), 10.17 (s, 1H),9.74 (s, 1H), 9.02 (t, J=5.9 Hz, 1H), 7.86-7.77 (m, 1H), 7.58-7.46 (m,4H), 7.45-7.37 (m, 2H), 6.73 (d, J=11.9 Hz, 3H), 6.33 (s, 1H), 5.13 (dd,J=13.2, 5.1 Hz, 1H), 4.50 (d, J=17.6 Hz, 1H), 4.41 (d, J=17.6 Hz, 1H),3.76 (s, 2H), 3.48 (s, 2H), 3.25-3.13 (m, 4H), 3.02-2.85 (m, 2H),2.66-2.57 (m, 1H), 2.45-2.31 (m, 1H), 2.14 (s, 6H), 2.04-2.02 (m, 1H),1.06 (t, J=7.2 Hz, 3H), 0.91 (d, J=6.9 Hz, 6H).

ESMS calculated for C₄₇H₄₉N₉O₉: 883.37; Found: 884.1 (M+H)⁺.

SDC-TRAP-0178 was tested in the HER2 degredation assays described inExample 12. There results are set forth in the table below.

SDC-TRAP-0178 HER2 Degradation Assay

SDC-TRAP# HER2 Degradation IC50 (nM) SDC-TRAP-0178 91 nM

SDC-TRAP-0178 Mouse Plasma Stability Assay

The percentage of a 10 μmole (μM) intravenous dose of SDC-TRAP-0178remaining in plasma of a mouse after 1 hour was determined by theprotocol set forth in Example 16:

Compound ID % Remaining (1 h, 10 μM) SDC-TRAP-0178 82.0%

SDC-TRAP-0178 Tissue Distribution

Tissue distribution of SDC-TRAP-0178 in plasma and tumor was determinedfollowing the protocol set forth in Example 14. Data therefrom are setforth in the table below:

Tumor Conc. Plasma Conc. (μM) (nmol/g of tissue) Tumor/Plasma Ratio SDC-SDC- SDC- SDC- SDC- SDC- Analyte TRAP- TRAP- TRAP- TRAP- TRAP- TRAP-Target 0178 0183 Lenalidomide 0178 0183 Lenalidomide 0178 0183Lenalidomide Time (h) 0.083 918 N/A 1.39 16.4 0.320 0.623 0.0179 — 0.4491 217 N/A 0.963 12.8 0.316 0.629 0.0589 — 0.653 6 4.51 N/A 0.00447 7.170.418 0.0532 1.59 — 11.9 24 0.0280 N/A BQL 2.81 0.556 BQL 100 — — 480.241 N/A BQL 1.01 0.508 BQL — — —

Determination of Cytotoxicity of Additional SDC-TRAP Molecules

The cytotoxicity of additional SDC-TRAP molecules was determined inBT-474, SW780 and RT-112 cancer cell lines. Cytotoxicity was determinedfollowing the protocol set forth in Example 13. Results are presented inthe table below.

Cytotoxicity (IC₅₀, nM) Compounds Payload BT-474 RT-112 SW-780SDC-TRAP-0069 Bendamustine 914 909 1,342 SDC-TRAP-0211 Bendamustine 249110 2,341 SDC-TRAP-0098 VDA 41 22 257 SDC-TRAP-0198 Doxorubicin 786297 >10,000 SDC-TRAP-0199 Doxorubicin 29 29 2,299 SDC-TRAP-0219Doxorubicin >10,000 973 >10,000 SDC-TRAP-0200 Doxorubicin 32 16 651SDC-TRAP-0068 Pemetrexed 70 74 202 fragment SDC-TRAP-0093 Pemetrexed1,540 1287 >10,000 fragment SDC-TRAP-0117 Vorinostat 452 152 284SDC-TRAP-0201 SN-38 1406 72 1,097 SDC-TRAP-0204 SN-38 8062 1314 >10,000SDC-TRAP-0046 SN-38 205 20 489 SDC-TRAP-0063 SN-38 320 83 261SDC-TRAP-0171 Lenalidomide 58 20 275 SDC-TRAP-0178 Lenalidomide 3729 >10,000 SDC-TRAP-0196 Lenalidomide 17 31 >10,000Lenalidomide >10,000 >10,000 >10,000 (17-AAG) 42 44 161 (SN-38) >10,000<10 38

Example 9

Determination of IC₅₀ by Assessing the Effects of Various SDC-TRAPs onTumor Shrinkage

H3122 cells were seeded into in 96-well plates at 7,500 cells/90μL/well, and were incubated for 24 hours. 14 SDC-TRAPs, plus ganetespibas a control, were serially diluted in dimethylsulfoxide (DMSO) intoeach of six wells of each 96-well plate according to the graphic below,where each cell represents a well in the plate.

To each well of plates #1 and 3 (continuous plates), 145 μL of media wasadded, and the cells were incubated. The wells of plates #2 and 4(pulsed plates) were incubated for 1 hour, then the wells were rinsed 2×with fresh media to remove the conjugate, and 145 μL of media was thenadded to each washed well. IC₅₀ was determined visually under amicroscope after 48 hours and 72 hours drug-exposure. Also at the 72hour time point, 50 μL of the cell culture supernatant was mixed with 50μL of CellTiter-Glo and the luminescence was determined, from which anIC₅₀ for each conjugate was calculated.

The data demonstrating the tumor effect of these SDC-TRAPs are set forthin FIGS. 4-8.

Example 10 IC₅₀ of Continuous and Pulsed Exposure to SDC-TRAPs

IC₅₀ toxicity was determined for 72 hour continuous exposure to 14SDC-TRAPs run in triplicate, and for duplicate pulse exposure (1 hour“pulse” exposure to conjugate compound, followed by 72 hour incubationin conjugate-free media) using H3211 cells, according to the protocolset forth in Example 9. The experimental data are set forth in the tablebelow.

1 h- pulse/ 1 h-pulse/ 71 h- 72 h- 72 h- 72 h- 71 h-compound compoundcompound continuous continuous continuous free free H 3211 NSLC cellsSDC-    12>    12>    12> 82 88 (7.5 × 10{circumflex over ( )}3cells/well), TRAP-0223 plate #1 (continuous),SDC- >3000    >3000    >3000    >3000 >3000 #2 (pulse), n = 1 TRAP-0003SDC-  22  60  40 624 1748 TRAP-0004SDC- >3000    >3000    >3000    >3000 >3000 TRAP-0005 SDC-  21  49 27 >3000 756 TRAP-0006 SDC- 144 327 232 291 >3000 TRAP-0010 SDC- 7962227  796 >3000 >3000 TRAP-0015SDC- >3000    >3000    >3000    >3000 >3000 TRAP-0017 SDC- 287 839735 >3000 >3000 TRAP-0018 SDC- 209 713 258 >3000 >3000 TRAP-0019 SDC-587 2615  2009  >3000 >3000 TRAP-0020 SDC- 431 817 902 >3000 >3000TRAP-0021 SDC- 193 823 460 >3000 >3000 TRAP-0022 SDC-  59 239113 >3000 >3000 TRAP-0023 SDC- 76 118 104 697 2211 TRAP-0024 H 3211 NSLCSDC- >12    12>    12> 49 116 cells TRAP-0223 (7.5 × 10{circumflex over( )}3 SDC- 984 1743  1335  >3000 >3000 cells/well), TRAP-0027 plate #3(continuous), SDC- 468 1761  499 >3000 >3000 #4 (pulse), n = 1 TRAP-0028SDC-  79 191 106 >3000 >3000 TRAP-0029 SDC-  53  38  53 >3000 >3000TRAP-0030 SDC- 250 407 333 >3000 >3000 TRAP-0032 SDC- 587 1167 2046  >3000 >3000 TRAP-0034 SDC- 260 830 787 >3000 >3000 TRAP-0035 SDC-139 265  96 >3000 >3000 TRAP-0036SDC- >3000    >3000    >3000    >3000 >3000 TRAP-0224 SDC-    12>    12>   12> 108 1481 TRAP-0225 SDC- 152 292 232 1089 2901 TRAP-0226SDC- >3000    >3000    >3000    >3000 >3000 TRAP-0227SDC- >3000    >3000    >3000    >3000 >3000 TRAP-0228 SDC- >12    12>   12> 60 111 TRAP-0223

Example 11 Hsp90^(α) Binding Assay Protocol

An Hsp90^(α) fluorescence assay kit from BPS Bioscience (Cat #50294)containing Hsp90 recombinant enzyme, FITC-labeled geldanamycin, assaybuffer and a low binding 384-well plate was used to assay Hsp90^(α)binding. Dithiothreitol (DTT) (Cat #D0643) and bovine serum albumin(BSA) (Cat #A2153) were obtained from Sigma-Aldrich. Fluorescencepolarization was measured using a PHERAstar microplate reader (BMGLABTECH GmbH, Ortenberg, Germany.)

The compounds were diluted to 1 mM in DMSO and loaded into a compounddilution plate to make 3-fold dilutions yielding a total of 8concentrations. 1 μL of compound was transferred from the dilution plateto the low binding assay plate provided in the assay kit. 5 mL ofHsp90^(α) binding solution was prepared having a final concentration of7 ng/μL Hsp90^(α), 5 nM FITC-labeled geldanamycin, 2 mM DTT and 0.1mg/mL BSA. 49 μL of binding solution was added to each microplate well,incubated at room temperature for 1 hour, then read using the PHERAstarmicroplate reader. The high control sample contained no compound plusHsp90^(α); the low control sample contained no compound and noHsp90^(α). Percent inhibition was calculated using high control as 100%and low control as 0% inhibition. The IC₅₀ was calculated using GraphPadPrism 4 software.

Example 12

HER2 Degradation Assay with BT-474 Cell Line

HER2 has emerged as a key target for anticancer drugs due to itsintrinsic involvement in the phosphatidylinositol-3-kinase-Akt/proteinkinase B (PI3K-Akt) and the mitogen-activated protein kinase (MAPK)pathways, both of which suppress apoptosis and promote tumor cellsurvival, gene transcription, angiogenesis, cellular proliferation,migration, mitosis, and differentiation. The degradation of HER2 is ameasure of efficacy of anticancer therapeutics that target Hsp90.Accordingly, the SDC-TRAP molecules of the invention that comprise abinding moiety that binds Hsp90 were tested in the following HER2degradation assay.

BT-474 cells (human breast cancer cell line ATCC HTB-20) were obtainedfrom ATCC and seeded into 12-well tissue culture plates at 0.2×10⁶/1.8mL/well. The cells were incubated for more than 6 hours at 37° C. inDMEM+10% FBS, +1% P/S, +1.5 g/L sodium bicarbonate. Each test compoundwas titrated in 4-fold dilutions from 5 μM to 78 nM with DMSO and 200 μLof the titration was added to each well of the cell plate. The DMSOfinal concentration was 0.2%. Cells were incubated overnight at 37° C.in 5% CO₂.

Media was decanted from the plate, cells were washed 1× in PBS. 400 μLtrypsin (EDTA) per well was added, and the cells were incubated for 2 to3 minutes. Cells were collected into FACS tubes containing 1 ml culturemedium to neutralize the trypsin and were centrifuged for 5 minutes at1200 rpm. Supernatant was decanted and the cells were resuspended in 5μL FITC (anti HER2/nu)/200 μL staining buffer (1×PBS+1% FBS+0.05% SodiumAzide)/tube. Controls were 5 μL IgG isotype control and staining bufferonly. Tubes were incubated for 30 minutes in the dark at roomtemperature. 1 mL staining buffer was added to each tube and the tubeswere centrifuged for 6 minutes at 1200 rpm. The supernatant was decantedand 300 μL staining buffer was added to each tube, which was store at 4°C. fpr FACS (cytometer) analysis. The cytometer readout was normalizedand the potency of each compound is evaluated with IC₅₀ calculated withXLfit™ software.

Example 13

Cytotoxicity Assay with Cancer Cell Lines

Cytotoxicity of SDC-TRAP molecules was determined in three cancer celllines. 5000 cells/100 μL/well of human breast cancer cell line BT-474(ATCC #HTB-20) and human urinary bladder cancer cell line SW780 (ATCC#CRL-2169) and 5000 cells/well of human urinary bladder cancer cell lineRT-112 were seeded into 96-well flat-bottom tissue cultures plates andincubated overnight at 37° C. in 5% CO₂. BT-474 and SW780 cells werecultured in DMEM+10% FBS, +1% P/S, +1.5 g/L sodium bicarbonate; RT-112cells were cultured in EMEM+10% FBS, +1% P/S. SDC-TRAP-0178 was titratedby 10-fold dilutions from 10 μM to 10 nM and added to the plate at 10μL/well. Final concentration of DMSO in the cell plate was 0.25%. Theplates were incubated for 72 hours at 37° C. in 5% CO₂. 80 μL ofCellTiter-Glo was added to each well, followed by room temperatureincubation in the dark for 15 minutes. Cell was determined byluminescence. IC₅₀ was calculated using XLfit™ software.

Example 14 Tissue Distribution Extraction Procedure for SDC-TRAP TumorSamples

SDC-TRAP molecules have the the ability to be specifically targeted todesired cells. For example, SDC-TRAP molecules can be targeted to tumorsand tumor cells in order to treat cancer. This example sets forth aprotocol to extract the SDC-TRAP molecules of the invention from tumorsamples.

A 150 ng/mL solution of SDC-TRAP-0002 in methanol was prepared using aninternal spiking solution (500 μg/mL SDC-TRAP-0002 in DMSO). Using the10 mM stock solutions of the SDC-TRAP molecule and its Hsp90i bindingmoiety and effector moiety in DMSO, spiking solutions were prepared at0.025, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 250, and 500 μM in DMSO. 5 μLof each spiking solution was added to a 96-deep well plate.

Quality control standards were prepared from 5 μL of 0.1, 1, and 10 μMcalibration standard spiking solution added in triplicate into 96-deepwell plate and adding 50 μL of matrix (plasma or homogenized tumor).

To prepare test samples, test plasma was diluted as needed using blankplasma. Tumor samples were pulverized in liquid nitrogen, weighed, andhomogenized in PBS at 5× volume to sample weight. 50 μL of unknownplasma or homogenized tumor sample was mixed with 5 μL of DMSO. Thesamples were extracted by precipitating calibration standards, QCstandards, and unknown samples with 200 μL of internal standardsolution. The samples were mixed by vortex at room temperature forapproximately 1.5 minutes, then centrifuge at 2-8° C. 150 μL ofsupernatant was collected and 25 μL of water added. Samples were mixedand analyzed by LC-MS/MS.

Example 15 SDC-TRAP-0063 Tissue Distribution Study in Mice

The following experiment was conducted in order to demonstrate theability of SDC-TRAP molecules to specifically target desired tissues. Anexemplary SDC-TRAP molecule, SDC-TRAP-0063, was administered to miceaccording to the protocol below and tissue samples were collected toevaluate tissue distribution.

Samples of plasma, heart and tumor were excised from a euthanized mouse,homogenized in PBS at 5 times tissue weight and diluted in 5 μL DMSO/50μL sample. Prior to analysis, 55 μL samples and calibration standardswere precipitated in 200 μL methanol in 96-well plates. Samples weremixed on a vortex mixer for 1.5 minutes at 1500 rpm at room temperature,then centrifuged at 4400 rpm for 10 minutes at 8° C. 150 μL of eachsupernatant was transferred to a well of a new 96-well plate, and 25 μLof water was added and mixed with the sample. The samples were analyzedby LCMS/MS using a Phenomenex Kinetex 2.6 μm C18 100 A, 30×2.1 mm columnat 0.5 mL/minute for 3.5 minutes with a TIS detector. For the analysisof samples from female SCID mice, a gradient of solvent A (water/0.1%formic acid) and B (acetonitrile/0.1% formic acid) was used as in TableA below. The solvent gradient used to analyze the tissues from male SDand CD-1 mice is shown in Table B below.

TABLE A Time (min) A B 0 80 20 1.7 5 95 2 5 95 2.1 80 20 3.5 80 20

TABLE B Time (min) A B 0 95 5 1.7 5 95 2 5 95 2.1 95 5 3.5 95 5

The distribution of SDC-TRAP-0063 and its expected degradants, DP-1,(ganetespib) and effector moiety SN-38 (irinotecan) in plasma, tumor andheart of female SCID mice at the illustrated time points followinginjection are shown in the tables below and in FIG. 9. Similar data werecollected from male SD mice (FIG. 10) and male CD-1 mice (FIG. 11.)Tabular data are not shown. In each case, data collected over 48 hourspost-treatment indicate that binding moiety and effector moietyaccumulate and persist in tumor, but rapidly diminish in plasma andheart, demonstrating the efficacy of the SDC-TRAP molecules.

Compound SDC-TRAP-0063 ID Lot 1 Dose 50 mg/10 mL/kg Species Female SCIDMouse (H1975) Route IV Formulation DRD Appearance N/A Accuracy N/AAnalyte SDC-TRAP-0063 DP-1 SN-38 Target Time (h) Plasma Conc. (μM) 0.083526 0.0662 20.4 6 1.69 0.0397 0.0509 24 0.00675 0.0175 0.0240 48 BQL0.00793 0.00524 Time (h) Tumor Conc. (nmol/g of tissue) 0.083 6.430.00758 1.47 6 1.61 0.111 0.730 24 0.203 0.404 0.618 48 0.0188 1.060.296 Time (h) Heart Conc. (nmol/g of tissue) 0.083 79.1 0.0271 0.927 60.536 0.207 BQL 24 BQL 0.0855 BQL 48 BQL 0.0238 BQL Time (h)Tumor/Plasma Ratio 0.083 0.0122 0.114 0.0721 6 0.958 2.79 14.3 24 30.123.1 25.8 48 — 134 56.4 Time (h) Heart/Plasma Ratio 0.083 0.151 0.4090.0454 6 0.318 5.21 — 24 — 4.90 — 48 — 3.00 —

The tissue distribution of SDC-TRAP-0056 and SDC-TRAP-0052 as well asSN-38 and irinotecan was evaluated in female SCID mice as set forthabove for SDC-TRAP-0063, DP-1 and SN-38. In each case, the datademonstrate that SDC-TRAP molecule and the effector moiety accumulateand persist in tumor, but rapidly diminish from the plasma,demonstrating the efficacy of the SDC-TRAP molecules. The data is shownin the table below.

Compound ID SDC-TRAP-0046 SDC-TRAP- Irinotecan 0052 Lot 2 1 RCN-102 Dose50 mg/10 mL/kg 25 mg/10 mL/kg 24 mg/10 mL/kg Species Female SCID Mouse(H1975) Route IV IV IV Formulation DRD DRD DRD Appearance Clear ClearClear Accuracy 81.6% 97.2% 97.1% Analyte Target SDC-TRAP- SDC-TRAP-SN-38 SDC-TRAP- Irinotecan SN-38 0046 0052 0052 Time (h) Plasma Conc.(μM) 0.083 360 0.0782 2.29 — — — 6 5.88 0.0917 0.0773 58.7 2.24 1.42 122.37 0.0612 0.0389 — — — 24 0.0542 0.0364 0.00955 0.0223 BQL BQL 48 BQL0.0107 BQL — — — Time (h) Tumor Conc. (nmol/g of tissue) 0.083 6.94 BQL0.298 — — — 6 4.97 0.241 0.448 13.9 13.1 1.44 12 5.21 0.407 0.344 — — —24 2.19 1.71 1.01 5.33 0.0307 BQL 48 0.188 1.01 BQL — — — Time (h)Tumor/Plasma Ratio 0.083 0.0193 — 0.130 — — — 6 0.844 2.63 5.80 0.2365.82 1.01 12 2.20 6.65 8.83 — — — 24 40.3 46.9 105 238 — — 48 — 94.4 — —— —

Example 16 Plasma Stability Protocol for SDC-TRAP Compounds

150 ng/mL solution of SDC-TRAP-0002 in methanol was prepared using theinternal standard spiking solution. This solution was used toprecipitate all plasma samples in the study. 200 μL was pipetted into a96 deepwell plate over dry ice. 10 μL of 1 mM stock in DMSO was added toa 1.5 mL microfuge tube, then 990 μL of plasma. Samples were mixed byvortex, then 50 μL of each sample was added in triplicate to a 96-wellplate containing internal standard solution. This was designated the 0hour time point sample. 250 μL of the remaining plasma sample was addedto each of four 96 deepwell plates—one per time point. Samples wereincubated at 37° C. with gentle shaking for 0.25, 0.5, and 1 hour. Aftereach time point, one plate of each sample was removed from the shakerand placed on wet ice for approximately 2 minutes. 50 μL plasma aliquots(in triplicate) were added to the deepwell plate containing internalstandard solution. After the last time point was extracted, the 96deepwell plate was vortexed, then centrifuged at 2-8° C. 150 μL ofsupernatant was collected and 25 μL of water was added. Samples weremixed and analyzed by LC-MS/MS.

Example 17 SDC-TRAP Stability in Mouse Plasma

The stability of seven SDC-TRAP types in mouse plasma was measured asfollows. 990 μL mouse plasma aliquots from a common stock were spikedwith 10 μL of 1 mM stock of one of seven SDC-TRAP samples identified inthe table below. Each sample was mixed and divided into 250 μL aliquots,each representing time points 0, 15 minutes, 30 minutes or 1 hour. Atthe prescribed time point, 3×50 μL samples were each mixed with 200 μLof methanol containing internal standard and held on dry ice until alltime point samples were extracted. The samples collectively were vortexmixed for 1.5 minutes at 1500 rpm, then centrifuged at 4400 rpm for 10minutes at 8° C. 150 μL of each supernatant was transferred to a new96-well plate, 25 μL of water added and mixed, then each sample wasanalyzed by LCMS/MS as described in Example 16. The data collected atone hour are set forth in the table below.

% Remaining Compound ID (1 h) SDC-TRAP- 11.1% 0063 SDC-TRAP- 91.5% 0064SDC-TRAP- 74.7% 0172 SDC-TRAP- 72.4% 0180 SDC-TRAP- 18.0% 0184 SDC-TRAP-68.1% 0185 SDC-TRAP- 57.9% 0186

These and data taken at times 0, 15 minutes, 30 minutes and 1 hour arepresented graphically in FIG. 12. As indicated in FIG. 12, the SDC-TRAPmolecules of the invention are stable in mouse plasma.

Example 18 SDC-TRAP Stability in Mouse Plasma and Cell Culture Media

The stability of six SDC-TRAP molecules with a variety of bindingmoieties and a particular effector moiety (SN-38/irinotecan) in mouseplasma and cell culture media was assessed. Mouse plasma samples wereprepared according to Example 16. 98 μL of DMEM+10% FBS, +1% P/S, +1.5g/L sodium bicarbonate cell culture media was mixed with 2 μL of DMSOand aliquotted into 96-well plates at 250 μL per 0, 1, 2, and 18 hourtime point. Plasma samples were mixed at 150 rpm for the required timeand extracted and processed for analysis according to Example 16.

3×50 μL media samples in 96 were held in 96-well plates at −80° C. untilthe last time point was extracted. 200 μL of methanol containing IS wasadded and mixed by vortex at 1500 rpm for 1.5 minutes at roomtemperature. The samples were centrifuged at 4400 rpm for 10 minutes at8° C. 150 μL of supernatant was transferred to a new 96-well plate; 25μL of water was added to each well; and mixed and the samples wereanalyzed according to the procedure described in Example 16.

Mouse (10 μM) Mouse (10 μM) Media (5 μM) Media (5 μM) Media (5 μM) % % %% % Remaining Remaining Remaining Remaining Remaining SDC-TRAP-# 1 h(37° C.) § 1 h (37° C.)* 1 h (37° C.) § 1 h (37° C.)* 19 h (37° C.)SDC-TRAP- 44% 47% 43% 46% 29% 0029 SDC-TRAP- — 95% — 67% 6% 0037SDC-TRAP- — 61% — 50% 41% 0044 SDC-TRAP- 34% 45% 72% 77% 50% 0045SDC-TRAP- 50% 52% 62% 65% 37% 0046 SN-38 — 64% — 82% 52% *Double peaksobserved in parent chromatogram. Data calculated with sum of both peaks.

Example 19: SDC-TRAPs Comprising Vorinostat

SDC-TRAP-0117

N1-((4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazine-1-carbonyl)oxy)-N8-phenyloctanediamide

¹H NMR (400 MHz, DMSO-d₆) δ 11.91 (s, 1H), 11.40 (s, 1H), 9.83 (s, 1H),9.58 (s, 1H), 9.39 (s, 1H), 7.62-7.54 (m, 2H), 7.35-7.23 (m, 4H),7.18-7.10 (m, 2H), 7.05-6.96 (m, 1H), 6.78 (s, 1H), 6.26 (s, 1H), 3.48(s, 2H), 3.40 (s, 4H), 2.97 (p, J=6.9 Hz, 1H), 2.40-2.24 (m, 6H), 2.07(t, J=7.3 Hz, 2H), 1.54 (dt, J=22.8, 7.3 Hz, 4H), 1.36-1.25 (m, 4H),0.95 (d, J=6.9 Hz, 6H); ESMS calculated for C₃₇H₄₅N₇O₇: 699.34; Found:700.3 (M+H)⁺.

SDC-TRAP-0118

N1-((4-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)piperidine-1-carbonyl)oxy)-N8-phenyloctanediamide

¹H NMR (400 MHz, DMSO-d₆) δ 11.88 (s, 1H), 11.37 (s, 1H), 9.84 (s, 1H),9.53 (d, J=19.5 Hz, 2H), 7.58 (dt, J=7.3, 1.0 Hz, 2H), 7.52-7.39 (m,3H), 7.32-7.22 (m, 2H), 7.06-6.90 (m, 2H), 6.69 (s, 1H), 6.43 (d, J=3.1Hz, 1H), 6.23 (s, 1H), 4.22 (t, J=7.1 Hz, 2H), 3.91 (s, 2H), 2.95-2.80(m, 3H), 2.29 (t, J=7.4 Hz, 2H), 2.07 (t, J=7.3 Hz, 2H), 1.79-1.64 (m,4H), 1.54 (dt, J=24.2, 6.6 Hz, 5H), 1.43 (s, 1H), 1.37-1.25 (m, 4H),1.16 (q, J=12.3, 9.7 Hz, 4H), 0.80 (d, J=6.8 Hz, 6H); ESMS calculatedfor C₄₁H₄₉N₇O₇: 751.37; Found: 752.3 (M+H)⁺.

in vitro activity was determined for these compounds using the HER2degradation assay set forth herein:

HER2 degradation SDC-TRAP# IC₅₀ (nM) SDC-TRAP-0117 1095 SDC-TRAP-01182352

Example 20: SDC-TRAPs Comprising 5-FU

Exemplary Synthetic Protocol:

STEP-1: To a solution of 5-fluorouracil 1 (650 mg, 5 mmol) in anhydrousDMF (8 mL), triethylamine (100 mg, 1 mmol) was added while stirring.After 5 min, methyl acrylate 2 (1 g, 10 mmol) was added dropwise.Stirring was continued for 36 h. The solvent was evaporated underreduced pressure, and the residue was purified on chromatographic column(95:5 CH₂Cl₂/MeOH) to give compound 3 (860 mg, 75%).

STEP-2: A solution of compound 3 (800 mg, 3.47 mmol) in a mixture ofMeOH (4 mL) and 2N aqueous solution NaOH (3 mL) was heated for 4 h at60° C. The solvent was removed under reduced pressure, and the residuewas subjected to acidification to pH2, using a solution of 10% HCl,resulting in acid 4 as white crystals. ¹H NMR (400 MHz, DMSO-d₆) δ:12.43 (s, 1H); 11.78 (s, 1H); 8.06 (d, J=7.2 Hz, 1H); 3.82 (t, J=6.9 Hz,2H); 2.63 (t, J=6.9 Hz, 2H)

STEP-3: To a solution of acid 4 (42 mg, 0.2 mmol) and amine 5 (82 mg,0.2 mmol) in anhydrous DMF (4 mL) was added EDC (60 mg, 0.3 mmol) andHOBT (27 mg, 0.2 mmol). The reaction mixture was stirred at roomtemperature for 5 h. The reaction mixture was diluted with 5 mL waterand extracted with 100 mL of ethyl acetate. The organic phase was driedwith sodium sulfate, filtered and evaporated, followed by flashchromatography (hexane-ethyl acetate 1:1 and ethyl acetate-methanol98:2) to give SDC-TRAP-0049 (95 mg, 80%) as a white solid.

¹H NMR (400 MHz, DMSO-d₆) δ 11.94 (s, 1H), 11.75 (s, 1H), 9.62 (s, 1H),9.42 (s, 1H), 8.04 (d, J=6.9 Hz, 1H), 7.32-7.30 (m, 2H), 7.15-7.12 (m,2H), 6.77 (s, 1H), 6.27 (s, 1H), 3.82 (t, J=6.8 Hz, 2H), 3.54-3.33 (m,6H), 2.90 (ddt, J=13.9, 9.7, 5.3 Hz, 1H), 2.73-2.60 (m, 2H), 2.34-2.29(m, 4H), 0.94 (dd, J=11.8, 6.9 Hz, 6H); ESMS calculated for C₂₉H₃₂FN₇O₆:593.24; Found: 594.2 (M+H)⁺.

The following compounds were made in the same general manner as above:

SDC-TRAP-0051

N-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)-3-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N-methylpropanamide

¹H NMR (400 MHz, DMSO-d₆) δ 11.90 (s, 1H), 11.75 (s, 1H), 9.56 (s, 1H),9.47 (d, J=14.3 Hz, 1H), 8.04 (d, J=6.9 Hz, 1H), 7.54-7.35 (m, 3H), 6.95(td, J=8.9, 2.0 Hz, 1H), 6.74 (d, J=13.6 Hz, 1H), 6.47-6.40 (m, 1H),6.23 (d, J=4.1 Hz, 1H), 4.37 (t, J=6.0 Hz, 1H), 4.28 (t, J=6.5 Hz, 1H),3.82 (t, J=6.8 Hz, 1H), 3.60 (q, J=6.8 Hz, 3H), 3.54-3.33 (m, 6H), 2.90(ddt, J=13.9, 9.7, 5.3 Hz, 1H), 2.73-2.60 (m, 5H), 2.34 (t, J=6.7 Hz,1H), 0.84 (dd, J=11.8, 6.9 Hz, 6H); ESMS calculated for C₂₉H₃₀FN₇O₆:591.22; Found: 592.1 (M+H)⁺.

SDC-TRAP-0048

N-(2-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethoxy)ethyl)-3-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)propanamide

¹H NMR (400 MHz, DMSO-d₆) δ 11.88 (s, 1H), 11.77 (s, 1H), 9.56 (s, 1H),9.48 (s, 1H), 8.00 (t, J=5.6 Hz, 1H), 7.93 (d, J=6.8 Hz, 1H), 7.50 (d,J=8.7 Hz, 1H), 7.41 (t, J=2.1 Hz, 2H), 6.93 (dd, J=8.6, 2.1 Hz, 1H),6.73 (s, 1H), 6.43 (d, J=3.2 Hz, 1H), 6.23 (s, 1H), 4.31 (t, J=5.3 Hz,2H), 3.81 (t, J=6.6 Hz, 2H), 3.67 (t, J=5.4 Hz, 2H), 3.57 (s, 1H), 3.483.31 (m, 13H), 3.15 (q, J=5.6 Hz, 2H), 2.90 (p, J=6.8 Hz, 1H), 2.45 (t,J=6.7 Hz, 2H), 0.83 (d, J=6.9 Hz, 6H); ESMS calculated for C₃₀H₃₂FN₇O₇:621.23; Found: 622.2 (M+H)⁺.

SDC-TRAP-0050

N-(2-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethoxy)ethyl)-3-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N-methylpropanamide

¹H NMR (400 MHz, DMSO-d₆) δ 11.88 (s, 1H), 11.76 (s, 1H), 9.56 (s, 1H),9.49 (d, J=3.0 Hz, 1H), 8.03 (d, J=6.8 Hz, 1H), 7.49 (d, J=8.7 Hz, 1H),7.45-7.32 (m, 2H), 6.92 (dd, J=8.6, 2.1 Hz, 1H), 6.73 (d, J=1.6 Hz, 1H),6.41 (dd, J=13.7, 3.1 Hz, 1H), 6.23 (s, 1H), 4.32 (q, J=5.2 Hz, 2H),3.88 (s, 2H), 3.80 (td, J=6.9, 3.6 Hz, 2H), 3.71-3.63 (m, 2H), 3.47 (dd,J=19.9, 8.3 Hz, 7H), 2.90 (hept, J=7.0 Hz, 1H), 2.80 (s, 2H), 2.76-2.60(m, 4H), 0.84 (d, J=6.9 Hz, 6H); ESMS calculated for C₃₁H₃₄FN₇O₇:635.25; Found: 636.2 (M+H)⁺.

SDC-TRAP-0009

1-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)-3-(5-fluoro-2-oxo-1,2-dihydropyrimidin-4-yl)urea

¹H NMR (400 MHz, DMSO-d₆) δ 11.86 (s, 1H), 9.52 (s, 1H), 9.46 (d, J=4.8Hz, 1H), 8.10-7.82 (m, 2H), 7.59-7.39 (m, 3H), 6.95 (t, J=7.7 Hz, 1H),6.73 (d, J=9.6 Hz, 1H), 6.44 (dd, J=16.8, 3.3 Hz, 1H), 6.22 (s, 1H),4.31 (dt, J=12.6, 6.4 Hz, 2H), 3.57-3.48 (m, 2H), 2.90 (h, J=7.1 Hz,1H), 0.84 (t, J=7.8 Hz, 6H); ESMS calculated (C₂₆H₂₅FN₈O₅): 548.2;found: 549.1 (M+H).

SDC-TRAP-0025

2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl(5-fluoro-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate

¹H NMR (400 MHz, Methanol-d₄) δ 7.77 (d, J=5.3 Hz, 1H), 7.61 (d, J=8.6Hz, 1H), 7.51 (d, J=2.0 Hz, 1H), 7.42 (t, J=3.9 Hz, 1H), 7.07 (dd,J=8.7, 2.1 Hz, 1H), 6.51 (q, J=3.4 Hz, 2H), 6.26 (d, J=2.7 Hz, 1H),4.57-4.47 (m, 4H), 2.84 (q, J=6.8 Hz, 1H), 0.61 (d, J=6.8 Hz, 6H); ESMScalculated (C₂₆H₂₄FN₇O₆): 549.2; found: 550.2 (M+H).

SDC-TRAP-0013

N-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)-2-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide

¹H NMR (400 MHz, DMSO-d₆) δ 11.85 (s, 2H), 9.53 (s, 1H), 9.45 (s, 1H),8.34 (t, J=5.6 Hz, 1H), 7.96 (d, J=6.7 Hz, 1H), 7.51-7.38 (m, 3H), 6.95(dd, J=8.6, 2.1 Hz, 1H), 6.78 (s, 1H), 6.43 (d, J=3.1 Hz, 1H), 6.22 (s,1H), 4.23 (d, J=7.9 Hz, 3H), 3.46-3.34 (m, 2H), 3.35-3.26 (m, 1H),2.98-2.88 (m, 1H), 0.88 (d, J=6.9 Hz, 6H). ppm; ESMS calculated forC₂₇H₂₆FN₇O₆: 563.2; found: 563.9 (M+H+).

SDC-TRAP-0137

1-(2-(4-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)piperidin-1-yl)-2-oxoethyl)-5-fluoropyrimidine-2,4(1H,3H)-dione

¹H NMR (400 MHz, Chloroform-d) δ 7.57 (d, J=2.4 Hz, 1H), 7.44 (dt,J=6.5, 3.1 Hz, 1H), 7.40-7.28 (m, 3H), 7.19 (q, J=3.3 Hz, 1H), 7.12 (dq,J=8.6, 3.8, 3.0 Hz, 1H), 6.52 (q, J=3.3 Hz, 1H), 6.44-6.27 (m, 2H),4.74-4.35 (m, 2H), 4.34-4.16 (m, 2H), 4.09 (ddt, J=19.4, 7.6, 3.9 Hz,1H), 3.43-3.28 (m, 1H), 3.18-2.96 (m, 2H), 2.84 (qd, J=8.1, 5.3 Hz, 1H),2.63 (t, J=12.4 Hz, 1H), 1.93-1.68 (m, 4H), 1.45-1.06 (m, 3H), 0.48 (dt,J=6.4, 3.0 Hz, 6H). ppm; ESMS calculated for C₃₂H₃₄FN₇O₆: 631.3; found:632.2 (M+H⁺).

in vitro activity was determined for these compounds using the HER2degradation assay set forth herein:

HER2 degradation SDC-TRAP-# IC₅₀ (nM) SDC-TRAP-0049 >5000SDC-TRAP-0048 >5000 SDC-TRAP-0050 >5000 SDC-TRAP-0051 >5000SDC-TRAP-0013 >5000 SDC-TRAP-0137 >5000

Example 21: SDC-TRAPs Comprising Abiraterone

SDC-TRAP-0150

(3S,8R,9S,10R,13S,14S)-10,13-dimethyl-17-(pyridin-3-yl)-2,3,4,7,8,9,10,11,12,13,14,15-dodecahydro-1H-cyclopenta[a]phenanthren-3-yl4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazine-1-carboxylate

¹H NMR (400 MHz, DMSO-d₆) δ 11.94 (s, 1H), 9.61 (s, 1H), 9.41 (s, 1H),8.59 (dd, J=2.3, 0.9 Hz, 1H), 8.43 (dd, J=4.8, 1.6 Hz, 1H), 7.76 (dt,J=8.1, 1.9 Hz, 1H), 7.38-7.27 (m, 3H), 7.18-7.10 (m, 2H), 6.78 (s, 1H),6.26 (s, 1H), 6.12 (s, 1H), 5.38 (d, J=4.9 Hz, 1H), 4.34 (tt, J=10.8,4.8 Hz, 1H), 3.47 (s, 2H), 2.97 (p, J=6.9 Hz, 1H), 2.36-2.16 (m, 7H),2.05 (dt, J=15.2, 8.2 Hz, 3H), 1.82-1.46 (m, 8H), 1.40 (td, J=12.2, 5.0Hz, 1H), 1.03 (d, J=5.6 Hz, 8H), 0.95 (d, J=6.8 Hz, 6H). ESMS calculatedfor C₄₇H₅₆N₆O₅: 784.43; Found: 785.3 (M+H)⁺.

SDC-TRAP-0151

(3S,8R,9S,10R,13S,14S)-10,13-dimethyl-17-(pyridin-3-yl)-2,3,4,7,8,9,10,11,12,13,14,15-dodecahydro-1H-cyclopenta[a]phenanthren-3-yl(2-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethoxy)ethyl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 11.88 (s, 1H), 9.55 (s, 1H), 9.47 (s, 1H),8.60 (d, J=2.4 Hz, 1H), 8.44 (dd, J=4.7, 1.6 Hz, 1H), 7.77 (dt, J=8.2,1.9 Hz, 1H), 7.50 (d, J=8.7 Hz, 1H), 7.44-7.30 (m, 3H), 7.06 (q, J=6.4,5.7 Hz, 1H), 6.91 (dd, J=8.7, 2.0 Hz, 1H), 6.73 (s, 1H), 6.40 (d, J=3.1Hz, 1H), 6.22 (s, 1H), 6.12 (dd, J=3.3, 1.8 Hz, 1H), 5.38 (d, J=4.9 Hz,1H), 4.32 (q, J=5.8, 5.3 Hz, 3H), 3.67 (t, J=5.3 Hz, 2H), 3.08 (q, J=5.8Hz, 2H), 2.96-2.84 (m, 1H), 2.33-2.17 (m, 3H), 2.11-1.96 (m, 3H),1.87-1.35 (m, 8H), 1.12-1.00 (m, 8H), 0.83 (d, J=6.9 Hz, 6H); ESMScalculated for C₄₈H₅₆N₆O₆: 812.43; Found: 813.3 (M+H)⁺.

SDC-TRAP-0153

(3S,8R,9S,10R,13S,14S)-10,13-dimethyl-17-(pyridin-3-yl)-2,3,4,7,8,9,10,11,12,13,14,15-dodecahydro-1H-cyclopenta[a]phenanthren-3-yl4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperidine-1-carboxylate

¹H NMR (400 MHz, DMSO-d₆) δ 11.93 (s, 1H), 9.61 (s, 1H), 9.43 (s, 1H),8.59 (s, 1H), 8.43 (dd, J=4.8, 1.6 Hz, 1H), 7.76 (dt, J=8.2, 2.0 Hz,1H), 7.38-7.29 (m, 1H), 7.18 (d, J=8.6 Hz, 2H), 7.14-7.06 (m, 2H), 6.75(s, 1H), 6.27 (s, 1H), 6.12 (dd, J=3.1, 1.7 Hz, 1H), 5.38 (s, 1H), 4.33(tt, J=10.9, 4.7 Hz, 1H), 3.94 (d, J=12.6 Hz, 2H), 2.96 (p, J=6.8 Hz,1H), 2.67 (s, 2H), 2.37-2.16 (m, 3H), 2.04 (td, J=14.7, 13.8, 4.7 Hz,3H), 1.87-1.60 (m, 6H), 1.53 (d, J=12.9 Hz, 5H), 1.40 (td, J=12.2, 5.0Hz, 1H), 1.13-0.90 (m, 15H); ESMS calculated for C₄₈H₅₇N₅O₅: 783.44;Found: 784.5 (M+H)⁺.

in vitro activity was determined for these compounds using the HER2degradation assay set forth herein:

HER2 degradation SDC-TRAP-# IC₅₀ (nM) SDC-TRAP-0150 1407 SDC-TRAP-01511194 SDC-TRAP-0153 6336

Mouse Plasma Stability Data

SDC-TRAP-# % Remaining (1 h) SDC-TRAP-0150 103%

Example 22: SDC-TRAPs Comprising Bendamustine SDC-TRAP-02114-(5-(bis(2-chloroethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)-N-(2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)butanamide

A mixture of (5-aminoisoindolin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone (a, 0.1 mmol), bendamustine (b, 0.1 mmol) and HATU (0.1 mmol)in DMF (2 mL) was stirred at room temperature for 16 h. The mixture wasdiluted with 50 mL of water and extracted with 50 mL×2 EtOAc, and theorganic layers were combined, concentrated and purified by column toyield SDC-TRAP-0211 as a white solid (25 mg, 0.04 mmol).

¹H NMR (400 MHz, Chloroform-d) δ 7.62 (s, 1H), 7.41 (s, 1H), 7.28 (s,1H), 7.20 (t, J=9.3 Hz, 2H), 6.96 (d, J=2.3 Hz, 1H), 6.80 (dd, J=8.9,2.4 Hz, 1H), 6.38 (d, J=2.5 Hz, 1H), 5.00 (d, J=5.3 Hz, 4H), 3.77-3.68(m, 6H), 3.61 (t, J=6.7 Hz, 4H), 3.25 (p, J=6.9 Hz, 1H), 2.97 (t, J=6.8Hz, 2H), 2.49 (d, J=14.8 Hz, 4H), 2.20 (dq, J=20.9, 7.1 Hz, 2H),1.31-1.17 (m, 6H). ESMS calculated for C₃₄H₃₉Cl₂N₅O₄: 651.2; found:652.0 (M+H⁺).

SDC-TRAP-0039

4-(5-(bis(2-chloroethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)-N-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)-N-methylbutanamide

¹H NMR (400 MHz, DMSO-d₆) δ 11.85 (d, J=1.9 Hz, 1H), 9.61 (s, 1H), 9.58(s, 1H), 7.50-7.32 (m, 4H), 6.92-6.74 (m, 4H), 6.42 (s, 1H), 6.22 (d,J=1.6 Hz, 1H), 4.38-4.30 (m, 2H), 3.71-3.58 (m, 14H), 2.95-2.73 (m, 3H),2.40-2.35 (m, 2H), 1.90-1.98 (m, 2H), 0.84 (dd, J=6.9, 4.4 Hz, 6H); ESMScalculated for C₃₈H₄₄Cl₂N₈O₄: 746.29; Found: 747.3 (M+H)⁺.

SDC-TRAP-0040

4-(5-(bis(2-chloroethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)-N-(2-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethoxy)ethyl)-N-methylbutanamide

¹H NMR (400 MHz, DMSO-d₆) δ 11.86 (d, J=1.9 Hz, 1H), 9.60 (s, 1H), 9.55(s, 1H), 7.49-7.28 (m, 4H), 6.95-6.87 (m, 2H), 6.73-6.70 (m, 2H), 6.39(s, 1H), 6.24 (d, J=1.6 Hz, 1H), 4.30 (dt, J=16.3, 5.2 Hz, 2H),3.73-3.62 (m, 13H), 2.86-2.73 (m, 6H), 2.41-2.35 (m, 2H), 1.93 (dd,J=10.0, 5.1 Hz, 2H), 0.84 (dd, J=6.9, 4.4 Hz, 6H); ESMS calculated forC₄₀H₄₈Cl₂N₈O₅: 790.31; Found: 791.3 (M+H)⁺.

SDC-TRAP-0069

4-(5-(bis(2-chloroethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)-1-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)butan-1-one

¹H NMR (400 MHz, DMSO-d₆) δ 11.93 (s, 1H), 9.61 (s, 1H), 9.41 (s, 1H),7.31 (dd, =8.5, 4.6 Hz, 3H), 7.18-7.10 (m, 2H), 6.91 (d, J=2.3 Hz, 1H),6.82-6.74 (m, 2H), 6.27 (s, 1H), 3.71-.3.68 (m, 10H), 3.65 (s, 3H), 3.43(dd, J=12.5, 7.2 Hz, 6H), 2.96 (h, J=6.9 Hz, 1H), 2.82 (t, J=7.4 Hz,2H), 2.44 (t, J=7.2 Hz, 2H), 2.31 (dt, J=26.0, 5.1 Hz, 4H), 1.97 (d,J=11.4 Hz, 2H), 0.94 (d, J=6.8 Hz, 6H); ESMS calculated forC₃₈H₄₆Cl₂N₈O₄: 748.30; Found: 749.1 (M+H)⁺.

In vitro activity was determined for these compounds using the HER2degradation assay set forth herein:

HER2 degradation SDC-TRAP-# IC₅₀ (nM) SDC-TRAP-0039 2925 SDC-TRAP-00404741 SDC-TRAP-0069 1232 SDC-TRAP-0211 289

Example 23: SDC-TRAPs Comprising Crizotinib SDC-TRAP-0134 preparation:(R)-4-(4-((4-(4-(4-(6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carbonyl)piperidin-1-yl)methyl)phenyl)-5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4H-1,2,4-triazole-3-carboxamide

A mixture of1-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzyl)piperidine-4-carboxylicacid (a, 25 mg, 0.05 mmol), crizotinib (23 mg, 0.05 mmol), DMAP (0.1mmol) and T3P (0.10 mmol) in 5 mL THF was heated in a microwave reactorat 80° C. for 1 h. The mixture was diluted with 100 mL each of 1M NaHCO₃solution and EtOAc. The organic layer was separated, dried, concentratedand purified by column chromatography to give(R)-4-(4-((4-(4-(4-(6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carbonyl)piperidin-1-yl)methyl)phenyl)-5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4H-1,2,4-triazole-3-carboxamide(SDC-TRAP-0134, 20 mg) as white solid.

¹H-NMR (CDCl₃) δδ 7.7 (d, 1H, J=4), 7.5 (m, 4H), 7.4 (m, 1H), 7.3 (m,3H), 7.0 (t, 1H, J=8), 6.9 (d, 1H, J=$), 6.54 (s, 1H), 6.50 (s, 1H), 6.1(q, 1H, t=8), 4.95 (s, 2H), 4.8 (m, 1H), 4.4 (m, 1H), 4.1 (m, 1H), 3.57(s, 1H), 3.4 (m, 1H), 2.8 (m, 1H), 2.6 (m, 1H), 1.8-2.2 (m, 12H), 1.9(d, 3H, J=8), 1.7 (m, 1H), 1.2 (m, 6H), 0.7 (d, 6H, J=8) ppm; ESMScalculated for C₄₈H₅₃Cl₂FN₁₀O₅: 938.4; found: 939.4 (M+H⁺).

SDC-TRAP-0139:

(R)-4-(4-((2-(4-(4-(6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-y)piperidin-1-yl)-2-oxoethyl)(methyl)carbamoyl)phenyl)-5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4H-1,2,4-triazole-3-carboxamide

¹H-NMR (CDCl₃) δ 7.7 (m, 3H), 7.57 (s, 1H), 7.53 (s, 1H), 7.4 (m, 3H),7.3 (m, 1H), 7.0 (t, 1H, J=8), 6.89 (s, 1H), 6.51 (s, 1H), 6.45 (s, 1H),m 6.1 (t, 1H, J=8), 4.89 (s, 2H), 4.7 (m, 1H), 4.4 (m, 2H), 4.1 (m, 1H),3.4 (m, 2H), 3.2 (m, 2H), 2.9 (m, 2H), 2.2-2.4 (m, 2H), 2.1 (m, 2H), 1.9(d, 3H, J=8), 1.2 (m, 6H), 0.7 (d, 6H, J=8) ppm; ESMS calculated forC₄₅H₄₇Cl₂FN₁₀O₆: 912.3; found: 913.3 (M+H⁺).

SDC-TRAP-0138:

(R)-(4-(4-(6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidin-1-yl)(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenyl)piperazin-1-yl)methanone

To a mixture of crizotinib (22 mg, 0.05 mmol) and 4-nitrophenylcarbonochloridate (10 mg, 0.05 mmol) was added 2 mL CHCl₃ whereafter themixture was stirred for 1 h. Solvent was removed to yield crude(R)-4-nitrophenyl4-(4-(6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate(b, 0.05 mmol).

To the above crude solids was added a solution of4-(5-hydroxy-4-(4-(piperazin-1-yl)phenyl)-4H-1,2,4-triazol-3-yl)-6-isopropylbenzene-1,3-diol(c, 20 mg, 0.05 mmol) in DMF (2 mL), and the mixture was heated to 110°C. for 10 h. The mixture was diluted in 100 mL each of water and EtOAc.The organic layer was separated, dried, concentrated and purified bycolumn chromatography to give(R)-(4-(4-(6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)pyridin-3-yl)-1H-pyrazol-1-yl)piperidin-1-yl)(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenyl)piperazin-1-yl)methanone(SDC-TRAP-0138, 4 mg) as a white solid.

¹H-NMR (CD₃OD) δ 7.7 (m, 1H), 7.6 (m, 2H), 7.4 (m, 3H), 7.2 (m, 2H), 7.1(m, 3H), 6.9 (m, 1H), 6.53 (s, 1H), 6.48 (s, 1H), 6.1 (m, 1H), 4.3 (m,1H), 3.9 (m, 1H), 3.2-3.8 (m, 7H), 3.0 (m, 2H), 1.8-2.3 (m, 8H), 1.3(3H, d, J=8), 0.8 (d, 6H, J=8) ppm; ESMS calculated for C₄₃H₄₅Cl₂FN₁₀O₅:870.3; found: 871.3 (M+H⁺).

in vitro activity was determined for these compounds using the HER2degradation assay set forth herein:

HER2 IC₅₀ No SDC-TRAP-# (nM) 1 SDC-TRAP-0134  77 2 SDC-TRAP-0138 707 3SDC-TRAP-0139 1000-2000

Hsp90^(α) Binding Activity Data:

Binding No SDC-TRAP-# EC₅₀ (nM) 1 SDC-TRAP-0134 95.42 nM

Hsp90^(α) Binding Data:

SDC-TRAP-# EC₅₀ (nM) SDC-TRAP-0134 95.42 nM

Mouse Plasma Stability Data:

% SDC- Remaining TRAP-# (1 h) SDC- 89.9% TRAP-0143 SDC- 96.2% TRAP-0144

Example 24: SDC-TRAPs Comprising Doxorubicin

Exemplary Synthesis:

To a solution of Hsp90 inhibitor fragment 1 (102 mg, 0.2 mmol) inanhydrous DMF (6 mL) was added HATU (78 mg, 0.2 mmol) under nitrogen at0° C., followed by diisopropylamine (78 mg, 0.6 mmol). The reactionmixture was stirred at 0° C. for 15 min, followed by the addition ofdoxorubicin hydrochloride 2 (135 mg, 0.25 mmol), and stirring wascontinued for 18 h at room temperature. The reaction mixture was dilutedwith methylene chloride and washed with water and brine. The organicphase was dried with sodium sulfate, filtered and concentrated, leavinga dark red residue. The product was isolated using column chromatography(95:5 dichloromethane/methanol) to give SDC-TRAP-0142(ethyl-5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-((4-(((2S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)tetrahydro-2H-pyran-4-yl)carbamoyl)piperidin-1-yl)methyl)phenyl)-4H-1,2,4-triazole-3-carboxylate,115 mg, 55%) as a red solid.

¹H NMR (400 MHz, DMSO-d₆) δ 14.02 (s, 1H), 13.27 (s, 1H), 10.62 (s, 1H),9.76 (s, 1H), 8.93 (t, J=5.9 Hz, 1H), 7.90 (d, J=4.8 Hz, 2H), 7.64 (p,J=3.8 Hz, 1H), 7.44 (d, J=8.1 Hz, 1H), 7.35 (d, J=8.0 Hz, 2H), 7.27 (d,J=8.0 Hz, 2H), 6.55 (s, 1H), 6.33 (s, 1H), 5.44 (s, 1H), 5.22 (d, J=3.4Hz, 1H), 4.94 (t, J=4.4 Hz, 1H), 4.85 (t, J=5.9 Hz, 1H), 4.72 (d, J=5.8Hz, 1H), 4.57 (d, J=5.9 Hz, 2H), 4.16 (q, J=6.7 Hz, 1H), 4.08-3.93 (m,3H), 3.41 (d, J=17.4 Hz, 3H), 3.15 (p, J=7.0 Hz, 2H), 3.05-2.77 (m, 5H),2.24-2.06 (m, 3H), 1.95-1.79 (m, 3H), 1.60-1.36 (m, 5H), 1.15 (dd,J=23.9, 6.7 Hz, 2H), 1.02 (t, J=7.1 Hz, 3H), 0.77 (d, J=6.8 Hz, 6H).ESMS calculated for C₅₄H₅₉N₅O₁₆: 1033.40; Found: 1033.8 (M+H)⁺.

The following compounds were made in the same general manner as above:

1-(1-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzyl)piperidine-4-carbonyl)-N-((2S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)tetrahydro-2H-pyran-4-yl)piperidine-4-carboxamide

¹H NMR (400 MHz, DMSO-d₆) δ 14.04 (s, 1H), 13.28 (s, 1H), 10.61 (s, 1H),9.79 (s, 1H), 8.96 (t, J=5.8 Hz, 1H), 7.91 (d, J=4.8 Hz, 2H), 7.69-7.61(m, 1H), 7.55 (d, J=8.1 Hz, 1H), 7.36 (d, J=8.0 Hz, 2H), 7.28 (d, J=7.9Hz, 2H), 6.57 (s, 1H), 6.34 (s, 1H), 5.47 (s, 1H), 5.22 (d, J=3.4 Hz,1H), 4.96-4.83 (m, 2H), 4.77 (t, J=6.0 Hz, 1H), 4.57 (d, J=5.9 Hz, 2H),4.33-4.16 (m, 2H), 3.98 (s, 3H), 3.46 (s, 2H), 3.21-3.09 (m, 2H),3.05-2.84 (m, 4H), 2.82-2.39 (m, 2H), 2.24-2.08 (m, 2H), 1.85 (t, J=12.1Hz, 1H), 1.61 (s, 3H), 1.54 (s, 4H), 1.41-1.26 (m, 3H), 1.16-0.98 (m,8H), 0.79 (d, J=6.8 Hz, 6H); ESMS calculated for C₆₀H₆₉N₇O₁₆: 1143.48;Found: 1144.2 (M+H)⁺.

5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4-(4-(4-(((2S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)tetrahydro-2H-pyran-4-yl)carbamoyl)phenoxy)phenyl)-4H-1,2,4-triazole-3-carboxamide;ESMS calculated for C₅₄H₅₃N₅O₁₆: 1027.35; Found: 1028.2 (M+H)⁺.

5-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenoxy)piperidin-1-yl)-N-((2S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)tetrahydro-2H-pyran-4-yl)pyrazine-2-carboxamide;ESMS calculated for C₅₇H₆₀N₈O₁₆: 1112.41; Found: 1113.2 (M+H)⁺.

SDC-TRAP-0219

(E)-N′-(1-((2S,4S)-4-(((2R,4S,5S,6S)-4-amino-5-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl)-2-hydroxyethylidene)-3-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)propanehydrazide

ESMS calculated for C₄₉H₅₁N₇O₁₄: 961.35; Found: 962.2 (M+H)⁺.

in vitro activity was determined for these compounds using the HER2degradation assay set forth herein:

HER2 degradation SDC-TRAP-# IC₅₀ (nM) SDC-TRAP-0142 >10,000SDC-TRAP-0198 >10,000 SDC-TRAP-0199 >10,000 SDC-TRAP-0200 >10,000

Hsp90^(α) Binding Assay Data

SDC-TRAP-# EC₅₀ (nM) SDC-TRAP-0198 93.32 SDC-TRAP-0199 136.3SDC-TRAP-0200 252.6

Example 25: SDC-TRAPs Comprising Lenalidomide

Exemplary Synthesis:

STEP-1: To a stirred suspension of lenalidomide 1 (520 mg, 2 mmol) indry THF (70 mL) was added 4-nitrophenylchloroformate (605 mg, 3 mmol).The reaction mixture was refluxed for 2 h, concentrated to approximately40 mL and triturated with ethyl acetate to yield a white precipitate.The solid was collected by filtration and washed with ethyl acetate togive activated lenalidomide 2 (650 mg, 77%).

STEP-2: Diisopropylethylamine (33 mg, 0.25 mmol) was added to a stirredsolution of Hsp90 inhibitor fragment 3 (120 mg, 0.2 mmol) and theactivated lenalidomide 2 (86 mg, 0.2 mmol) in anhydrous DMF (5 mL). Thereaction mixture was stirred at room temperature for 18 h. The reactionmixture was diluted with water (5 mL) and extracted with ethyl acetate(100 mL). Organic phase was dried (sodium sulfate) filtered andevaporation, followed by flash chromatography (hexane-ethyl acetate 1:1and ethyl acetate-methanol 98:2) gave SDC-TRAP-0178 (95 mg, 53%) as awhite solid.

¹H NMR (400 MHz, DMSO-d₆) δ 11.02 (s, 1H), 10.22 (s, 1H), 10.17 (s, 1H),9.74 (s, 1H), 9.02 (t, J=5.9 Hz, 1H), 7.86-7.77 (m, 1H), 7.58-7.46 (m,4H), 7.45-7.37 (m, 2H), 6.73 (d, J=11.9 Hz, 3H), 6.33 (s, 1H), 5.13 (dd,J=13.2, 5.1 Hz, 1H), 4.50 (d, J=17.6 Hz, 1H), 4.41 (d, J=17.6 Hz, 1H),3.76 (s, 2H), 3.48 (s, 2H), 3.25-3.13 (m, 4H), 3.02-2.85 (m, 2H),2.66-2.57 (m, 1H), 2.45-2.31 (m, 1H), 2.14 (s, 6H), 2.04-2.02 (m, 1H),1.06 (t, J=7.2 Hz, 3H), 0.91 (d, J=6.9 Hz, 6H). ESMS calculated forC₄₇H₄₉N₉O₉: 883.37; Found: 884.1 (M+H)⁺.

SDC-TRAP-0105

1-(2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)ethyl)-3-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)-1-methylurea

¹H NMR (400 MHz, Chloroform-d) δ 7.69 (dd, J=8.9, 6.4 Hz, 1H), 7.49 (dp,J=6.6, 3.6 Hz, 3H), 7.42-7.22 (m, 4H), 6.43 (dd, J=40.6, 2.5 Hz, 1H),5.17 (dd, J=13.7, 5.6 Hz, 1H), 4.41 (d, J=19.5 Hz, 2H), 4.13 (tt, J=8.7,4.3 Hz, 1H), 3.35 (d, J=17.6 Hz, 2H), 3.00 (p, J=4.9, 4.0 Hz, 4H),2.93-2.31 (m, 11H), 2.21 (d, J=13.0 Hz, 1H), 2.12-1.99 (m, 2H), 1.28(qd, J=7.5, 2.9 Hz, 3H), 0.92 (td, J=10.3, 9.7, 4.7 Hz, 1H), 0.75 (td,J=7.2, 2.7 Hz, 6H). ppm; ESMS calculated for C₃₉H₄₅N₉O₇: 751.3; found:752.3 (M+H⁺).

SDC-TRAP-0108

4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenethyl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)piperidine-1-carboxamide

¹H NMR (400 MHz, Chloroform-d) δ 8.05-7.97 (m, 1H), 7.63 (ddd, J=12.2,7.1, 3.1 Hz, 1H), 7.53-7.39 (m, 1H), 7.37-7.30 (m, 1H), 7.27-7.19 (m,2H), 6.43 (d, J=29.7 Hz, 1H), 5.14 (td, J=12.9, 5.2 Hz, 1H), 4.58-4.29(m, 2H), 4.22-4.01 (m, 2H), 3.59 (s, 2H), 3.37 (dt, J=3.4, 1.7 Hz, 1H),3.10-2.65 (m, 6H), 2.53-2.11 (m, 2H), 1.85 (d, J=14.3 Hz, 2H), 1.62(tdd, J=18.4, 9.2, 5.3 Hz, 3H), 1.37-1.14 (m, 3H), 0.75 (d, J=6.8 Hz,6H). ppm; ESMS calculated for C₃₈H₄₁N₇O₇: 707.3; found: 708.2 (M+H⁺).

SDC-TRAP-0126

4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenyl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)butanamide

¹H NMR (400 MHz, Methanol-d₄) δ 7.76 (d, J=7.9 Hz, 1H), 7.70 (d, J=7.5Hz, 1H), 7.51 (d, J=7.8 Hz, 1H), 7.48 (s, 3H), 7.28-7.18 (m, 2H),7.09-7.02 (m, 2H), 6.55 (s, 1H), 6.37 (s, 1H), 5.16 (dd, J=13.3, 5.1 Hz,1H), 4.50 (s, 2H), 3.39 (s, 2H), 3.36 (p, J=1.6 Hz, 4H), 2.99 (p, J=6.8Hz, 2H), 2.93-2.82 (m, 2H), 2.64 (t, J=6.9 Hz, 2H), 2.55-2.33 (m, 1H),2.22 (dp, J=12.9, 4.4 Hz, 1H), 2.09 (dt, J=13.7, 6.7 Hz, 3H), 0.80 (d,J=6.9 Hz, 6H). ppm; ESMS calculated for C₃₈H₄₂N₈O₇: 722.3; found: 723.3(M+H⁺).

SDC-TRAP-0132

3-(2-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenyl)-N-methylacetamido)propyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

ESMS calculated for C₃₇H₃₉N₇O₉: 725.3; found: 726.2 (M+H⁺).

SDC-TRAP-0127

2-(2-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenyl)-N-methylacetamido)ethyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 11.90 (s, 1H), 11.00 (s, 1H), 9.75-9.28 (m,3H), 7.70 (d, J=20.2 Hz, 1H), 7.57-7.38 (m, 3H), 7.21 (d, J=8.1 Hz, 2H),7.15-7.05 (m, 2H), 6.82 (d, J=2.2 Hz, 1H), 6.25 (s, 1H), 5.12 (dd,J=13.3, 5.2 Hz, 1H), 4.55-4.11 (m, 4H), 3.89-3.48 (m, 4H), 3.07 (s, 1H),3.03-2.79 (m, 1H), 2.74-2.55 (m, 1H), 2.50 (s, 3H), 0.98 (dd, J=7.0, 5.2Hz, 6H). ppm; ESMS calculated for C₃₆H₃₇N₇O₉: 711.3; found: 712.1(M+H⁺).

SDC-TRAP-0133

2-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)-N-methylbenzamido)ethyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 11.01 (s, 1H), 10.21 (d, J=17.5 Hz, 1H),9.72 (s, 1H), 9.60 (s, 1H), 9.01 (t, J=5.9 Hz, 1H), 7.70 (d, J=36.6 Hz,1H), 7.57-7.28 (m, 6H), 6.71 (s, 1H), 6.32 (s, 1H), 5.12 (dd, J=13.2,5.1 Hz, 1H), 4.52-4.16 (m, 4H), 3.77 (s, 1H), 3.52 (s, 1H), 3.18 (qd,J=7.3, 4.7 Hz, 2H), 3.10-2.79 (m, 5H), 2.75-2.55 (m, 1H), 2.45-2.23 (m,1H), 2.12-1.91 (m, 1H), 1.05 (t, J=7.2 Hz, 3H), 0.88 (d, J=6.8 Hz, 6H).ppm; ESMS calculated for C₃₈H₄₀N₈O₉: 752.3; found: 753.3 (M+H⁺).

SDC-TRAP-0135

3-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)-N-methylbenzamido)propyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 11.01 (s, 1H), 10.18 (s, 1H), 9.71 (s, 1H),9.57 (s, 1H), 9.00 (t, J=5.9 Hz, 1H), 7.77 (s, 1H), 7.51-7.43 (m, 5H),7.41-7.34 (m, 2H), 6.73 (s, 1H), 6.32 (s, 1H), 5.12 (dd, J=13.3, 5.1 Hz,1H), 4.41 (q, J=17.1, 16.2 Hz, 2H), 4.19 (s, 2H), 3.58 (s, 2H), 3.31 (s,2H), 3.18 (s, 3H), 3.02-2.84 (m, 3H), 2.60 (dt, J=15.7, 3.3 Hz, 1H),2.34 (d, J=13.0 Hz, 2H), 1.05 (t, J=7.4 Hz, 3H), 0.90 (d, J=6.8 Hz, 6H).ppm; ESMS calculated for C₃₉H₄₂N₈O₉: 766.3; found: 767.3 (M+H⁺).

SDC-TRAP-0140

2-(1-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzoyl)piperidin-4-yl)ethyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 11.03 (s, 1H), 10.30 (s, 1H), 9.75 (s, 1H),9.54 (s, 1H), 9.01 (t, J=5.9 Hz, 1H), 7.77 (dt, J=7.7, 3.8 Hz, 1H),7.54-7.36 (m, 6H), 6.68 (s, 1H), 6.33 (s, 1H), 5.13 (dd, J=13.3, 5.1 Hz,1H), 4.40 (q, J=17.6 Hz, 3H), 4.17 (t, J=6.5 Hz, 2H), 3.56 (s, 1H),3.24-3.13 (m, 2H), 3.07 (s, 1H), 2.92 (ddd, J=17.1, 13.5, 5.8 Hz, 2H),2.78 (s, 1H), 2.67-2.57 (m, 1H), 2.35 (qd, J=13.2, 4.4 Hz, 1H),2.08-1.97 (m, 1H), 1.71 (m, 4H), 1.62 (q, J=6.6 Hz, 2H), 1.22 (d, J=13.2Hz, 2H), 1.06 (t, J=7.2 Hz, 3H), 0.88 (d, J=6.9 Hz, 6H). ppm; ESMScalculated for C₄₂H₄₆N₈O₉: 806.3; found: 807.3 (M+H⁺).

SDC-TRAP-0136

(1-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzoyl)piperidin-4-yl)methyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 10.88 (s, 1H), 10.16 (s, 1H), 9.60 (s, 1H),9.40 (s, 1H), 8.87 (t, J=5.8 Hz, 1H), 7.63 (dd, J=6.7, 2.4 Hz, 1H),7.39-7.22 (m, 6H), 6.53 (s, 1H), 6.19 (s, 1H), 4.99 (dd, J=13.2, 5.1 Hz,1H), 4.35-4.17 (m, 2H), 3.94-3.81 (m, 3H), 3.10-2.98 (m, 2H), 2.85-2.70(m, 2H), 2.67 (s, 1H), 2.51-2.42 (m, 1H), 1.93-1.81 (m, 4H), 1.52 (s,2H), 1.03 (t, J=7.1 Hz, 3H), 0.91 (t, J=7.2 Hz, 3H), 0.73 (d, J=6.9 Hz,6H). ppm; ESMS calculated for C₄₁H₄₄N₈O₉: 792.3; found: 793.2 (M+H⁺).

SDC-TRAP-0231

3-(1-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzyl)-N-methylpiperidine-4-carboxamido)propyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

¹H NMR (400 MHz, Chloroform-d) δ 7.88 (d, J=8.1 Hz, 1H), 7.61 (t, J=6.8Hz, 2H), 7.57-7.49 (m, 2H), 7.51-7.41 (m, 2H), 7.32 (d, J=8.3 Hz, 2H),6.57-6.40 (m, 2H), 5.19 (dd, J=13.2, 5.1 Hz, 1H), 4.55-4.31 (m, 2H),4.13 (td, J=6.2, 3.0 Hz, 2H), 3.71-3.46 (m, 5H), 3.46-3.30 (m, 3H), 3.08(s, 3H), 3.01-2.72 (m, 4H), 2.29-2.14 (m, 1H), 2.06 (dd, J=11.8, 6.7 Hz,2H), 1.87 (dp, J=13.0, 7.6, 6.9 Hz, 4H), 1.70 (d, J=13.3 Hz, 2H),1.41-1.12 (m, 6H), 0.71 (dd, J=13.5, 6.9 Hz, 6H). ppm; ESMS calculatedfor C₄₅H₅₃N₉O₉: 863.4; found: 864.3 (M+H⁺).

SDC-TRAP-0147

5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-((2-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-2-oxoethyl)(methyl)carbamoyl)phenyl)-N-ethyl-4H-1,2,4-triazole-3-carboxamide

ESMS calculated for C₃₇H₃₈N₈O₈: 722.3; found: 723.2 (M+H⁺).

SDC-TRAP-0165

5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-((3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-3-oxopropyl)(methyl)carbamoyl)phenyl)-N-(2,2,2-trifluoroethyl)-4H-1,2,4-triazole-3-carboxamide

ESMS calculated for C₃₈H₃₇F₃N₈O₈: 790.3; found: 791.1 (M+H⁺).

SDC-TRAP-0163

1-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzyl)-N-((2S)-1-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-3-methyl-1-oxobutan-2-yl)piperidine-4-carboxamide

¹H NMR (400 MHz, Methanol-d₄) δ 7.80 (ddd, J=26.0, 8.0, 1.0 Hz, 1H),7.70 (ddd, J=7.6, 4.3, 1.0 Hz, 1H), 7.59-7.43 (m, 3H), 7.41 (s, 1H),7.38-7.31 (m, 2H), 6.50 (s, 1H), 6.43 (s, 1H), 5.15 (ddd, J=13.3, 5.1,3.6 Hz, 1H), 4.60-4.22 (m, 3H), 3.63 (s, 2H), 3.43-3.28 (m, 3H),3.09-2.77 (m, 5H), 2.52-2.01 (m, 6H), 1.94-1.70 (m, 4H), 1.32-1.13 (m,4H), 1.03 (dd, J=12.4, 6.7 Hz, 6H), 0.98-0.83 (m, 1H), 0.75 (d, J=6.9Hz, 6H). ppm; ESMS calculated for C₄₅H₅₃N₉O₈: 847.4; found: 848.3(M+H⁺).

SDC-TRAP-0164

5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-((4-((2S)-2-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamoyl)pyrrolidine-1-carbonyl)piperidin-1-yl)methyl)phenyl)-N-ethyl-4H-1,2,4-triazole-3-carboxamide

To a mixture of1-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzyl)piperidine-4-carboxylicacid (a, 0.90 mmol),(2S)—N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)pyrrolidine-2-carboxamide hydrochloride (b, 0.80 mmol) and HATU (1.0mmol) in DMF (10 mL) at room temperature was added DIPEA (3.0 mmol) andthe mixture was stirred at room temperature for 16 h. The mixture wasadded to a solution of NaHCO₃ (200 mL, 0.1M) and stirred for 30 minbefore filtering. The yellow filter cake was purified by column to yieldSDC-TRAP-0164 as a white solid (0.25 g, 0.29 mmol).

¹H NMR (400 MHz, DMSO-d₆) δ 11.06 (d, J=6.8 Hz, 1H), 10.69-10.60 (m,1H), 9.90 (s, 1H), 9.77 (s, 1H), 8.97 (t, J=5.9 Hz, 1H), 7.81-7.72 (m,1H), 7.60-7.46 (m, 2H), 7.42-7.27 (m, 4H), 6.57 (d, J=9.4 Hz, 1H), 6.34(s, 1H), 5.19-5.11 (m, 1H), 4.47 (d, J=8.3 Hz, 1H), 4.33 (t, J=12.4 Hz,2H), 3.68 (s, 1H), 3.61 (s, 1H), 3.49 (s, 2H), 3.21-3.13 (m, 2H), 2.90(d, J=18.7 Hz, 5H), 2.63 (s, 1H), 2.00 (s, 7H), 1.67 (s, 2H), 1.58 (s,3H), 1.03 (td, J=7.2, 3.1 Hz, 4H), 0.79 (ddd, J=17.0, 6.9, 2.3 Hz, 6H).ppm; ESMS calculated for C₄₅H₅₁N₉O₈: 845.4; found: 846.2 (M+H⁺).

SDC-TRAP-0166

5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(((2S)-1-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-1-oxopropan-2-yl)carbamoyl)phenyl)-N-ethyl-4H-1,2,4-triazole-3-carboxamide

¹H NMR (400 MHz, Chloroform-d) δ 8.09-7.98 (m, 2H), 7.92-7.76 (m, 1H),7.71 (dd, J=7.6, 2.4 Hz, 1H), 7.56-7.39 (m, 3H), 6.40 (dd, J=5.6, 1.5Hz, 2H), 5.17 (ddd, J=13.5, 5.2, 1.7 Hz, 1H), 4.93-4.75 (m, 1H),4.58-4.28 (m, 2H), 3.49-3.30 (m, 3H), 3.30-3.10 (m, 5H), 2.88 (dddd,J=26.5, 12.7, 6.1, 2.9 Hz, 3H), 2.53-2.33 (m, 1H), 2.32-2.08 (m, 1H),1.70-1.53 (m, 3H), 1.34-1.11 (m, 4H), 0.72 (dd, J=6.9, 3.6 Hz, 6H). ppm;ESMS calculated for C₃₇H₃₈N₈O₈: 722.3; found: 723.1 (M+H⁺).

SDC-TRAP-0188

5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(((2S)-1-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamoyl)phenyl)-N-ethyl-4H-1,2,4-triazole-3-carboxamide

¹H NMR (400 MHz, Methanol-d₄) δ 8.07 (ddd, J=8.9, 4.5, 2.1 Hz, 2H),7.90-7.64 (m, 2H), 7.58-7.41 (m, 3H), 6.46-6.28 (m, 2H), 5.17 (dd,J=13.3, 5.1 Hz, 1H), 4.67-4.35 (m, 3H), 3.45-3.26 (m, 4H), 3.04-2.67 (m,3H), 2.52-2.14 (m, 3H), 1.58 (dq, J=19.9, 7.5 Hz, 1H), 1.30-1.17 (m,5H), 1.18-1.03 (m, 5H), 1.04-0.90 (m, 1H), 0.72 (dt, J=7.1, 1.4 Hz, 6H).ppm; ESMS calculated for C₃₉H₄₂N₈O₈: 750.3; found: 751.1 (M+H⁺).

SDC-TRAP-0189

5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(((2S)-1-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)phenyl)-N-ethyl-4H-1,2,4-triazole-3-carboxamide

¹H NMR (400 MHz, Chloroform-d) δ 8.13-8.01 (m, 2H), 7.95-7.77 (m, 1H),7.74-7.63 (m, 1H), 7.56-7.39 (m, 3H), 6.41 (d, J=2.0 Hz, 1H), 6.35 (d,J=5.0 Hz, 1H), 5.17 (ddd, J=13.3, 5.1, 2.2 Hz, 1H), 5.01-4.78 (m, 1H),4.59-4.26 (m, 2H), 3.47-3.25 (m, 4H), 2.98-2.79 (m, 3H), 2.53-2.11 (m,2H), 1.91-1.67 (m, 3H), 1.24 (dt, J=17.9, 7.2 Hz, 4H), 1.08-0.95 (m,6H), 0.70 (ddd, J=7.0, 4.2, 1.3 Hz, 6H). ppm; ESMS calculated forC₄₀H₄₄N₈O₈: 764.3; found: 765.1 (M+H⁺).

SDC-TRAP-0190

5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-((2S)-2-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamoyl)pyrrolidine-1-carbonyl)phenyl)-N-ethyl-4H-1,2,4-triazole-3-carboxamide

¹H NMR (400 MHz, DMSO-d₆) δ 11.04 (s, 1H), 10.20 (d, J=3.7 Hz, 1H),10.03 (d, J=3.1 Hz, 1H), 9.72 (s, 1H), 9.03 (t, J=5.9 Hz, 1H), 7.80 (dd,J=7.6, 1.6 Hz, 1H), 7.69-7.58 (m, 2H), 7.60-7.47 (m, 2H), 7.41 (d, J=8.0Hz, 3H), 6.72 (s, 1H), 6.31 (d, J=1.3 Hz, 1H), 5.15 (dd, J=13.3, 5.1 Hz,1H), 4.66 (t, J=6.5 Hz, 1H), 4.50-4.29 (m, 2H), 3.56 (ddd, J=22.5, 9.7,5.7 Hz, 2H), 3.19 (p, J=6.8 Hz, 2H), 2.92 (qt, J=14.8, 7.4 Hz, 3H), 2.61(d, J=17.0 Hz, 1H), 2.35 (t, J=11.7 Hz, 3H), 2.15-1.80 (m, 4H), 1.06 (t,J=7.2 Hz, 3H), 0.90 (dd, J=7.3, 2.1 Hz, 6H). ppm; ESMS calculated forC₃₉H₄₀N₈O₈: 748.3; found: 749.1 (M+H⁺).

SDC-TRAP-0191

5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(4-(((2S)-1-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)phenoxy)phenyl)-N-ethyl-4H-1,2,4-triazole-3-carboxamide

¹H NMR (400 MHz, Methanol-d₄) δ 7.98-7.80 (m, 4H), 7.68 (ddd, J=7.7,5.3, 1.0 Hz, 1H), 7.48 (td, J=7.8, 3.4 Hz, 1H), 7.36 (d, J=6.9 Hz, 1H),7.24-7.13 (m, 4H), 6.55 (s, 1H), 6.45 (s, 1H), 5.16 (ddd, J=13.3, 5.1,1.8 Hz, 1H), 4.86 (ddp, J=8.7, 5.2, 2.5 Hz, 1H), 4.64-4.23 (m, 2H),3.49-3.27 (m, 3H), 3.04 (p, J=6.9 Hz, 1H), 2.85 (ddt, J=9.4, 5.1, 2.3Hz, 2H), 2.51-2.29 (m, 1H), 2.20 (ddd, J=13.5, 6.9, 3.7 Hz, 1H),1.89-1.74 (m, 3H), 1.25 (dt, J=13.4, 7.2 Hz, 5H), 1.12-1.00 (m, 6H),1.00-0.91 (m, 1H), 0.87 (d, J=6.9 Hz, 6H). ppm; ESMS calculated forC₄₆H₄₈N₈O₉: 856.4; found: 857.1 (M+H⁺).

SDC-TRAP-0192

5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(4-((2S)-2-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamoyl)pyrrolidine-1-carbonyl)phenoxy)phenyl)-N-ethyl-4H-1,2,4-triazole-3-carboxamide

¹H NMR (400 MHz, Methanol-d₄) δ 7.94 (ddd, J=25.0, 8.1, 1.0 Hz, 1H),7.81 (dt, J=8.3, 4.1 Hz, 1H), 7.72-7.58 (m, 3H), 7.48 (td, J=7.8, 6.2Hz, 1H), 7.42-7.30 (m, 1H), 7.23-7.11 (m, 4H), 6.54 (d, J=1.7 Hz, 1H),6.44 (s, 1H), 5.14 (dd, J=13.3, 5.1 Hz, 1H), 4.87 (dt, J=8.1, 5.3 Hz,1H), 4.56-4.33 (m, 2H), 3.75-3.65 (m, 3H), 3.52-3.29 (m, 4H), 3.03 (p,J=6.8 Hz, 1H), 2.83 (ddd, J=10.6, 5.5, 2.8 Hz, 2H), 2.53-2.09 (m, 7H),1.97 (dtd, J=15.5, 8.2, 7.2, 4.7 Hz, 1H), 1.25 (dt, J=13.5, 7.2 Hz, 4H),0.87 (d, J=6.9 Hz, 6H). ppm; ESMS calculated for C₄₅H₄₄N₈O₉: 840.3;found: 841.1 (M+H⁺).

SDC-TRAP-0193

1-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzyl)-N-((2S)-1-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-4-methyl-1-oxopentan-2-yl)piperidine-4-carboxamide

¹H NMR (400 MHz, Chloroform-d) δ 7.93-7.83 (m, 1H), 7.68 (d, J=7.5 Hz,1H), 7.62-7.41 (m, 4H), 7.32 (dd, J=8.2, 2.7 Hz, 2H), 6.51-6.45 (m, 1H),6.43 (d, J=1.8 Hz, 1H), 5.16 (ddd, J=13.9, 9.4, 5.1 Hz, 1H), 4.67-4.52(m, 1H), 4.53-4.20 (m, 2H), 3.68-3.49 (m, 2H), 3.46-3.28 (m, 3H),3.07-2.72 (m, 6H), 2.35-2.25 (m, 4H), 2.05 (d, J=6.5 Hz, 1H), 1.91-1.53(m, 6H), 1.34-1.14 (m, 6H), 1.05-0.92 (m, 6H), 0.71 (dt, J=6.9, 2.9 Hz,6H). ppm; ESMS calculated for C₄₆H₅₅N₉O₈: 861.4; found: 862.2 (M+H⁺).

SDC-TRAP-0122

2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 11.87 (s, 1H), 11.02 (s, 1H), 9.56 (d,J=14.1 Hz, 2H), 9.46 (s, 1H), 7.65 (s, 1H), 7.54 (d, J=8.7 Hz, 1H),7.52-7.39 (m, 4H), 6.95 (dd, J=8.7, 2.0 Hz, 1H), 6.74 (d, J=1.7 Hz, 1H),6.46 (d, J=3.1 Hz, 1H), 6.21 (s, 1H), 5.11 (dd, J=13.4, 5.0 Hz, 1H),4.49 (t, J=5.2 Hz, 2H), 4.44-4.25 (m, 4H), 2.84-2.85 (m, 2H), 2.65-2.56(m, 1H), 2.33 (td, J=13.4, 8.7 Hz, 1H), 2.03-1.95 (m, 1H), 0.83 (dd,J=7.1, 1.7 Hz, 6H); ESMS calculated (C₃₅H₃₃N₇O₈): 679.2; found: 680.2(M+H).

SDC-TRAP-0123

1-(1-(4-(3-(2,4-Dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzyl)piperidine-4-carbonyl)piperidin-4-yl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 11.01 (s, 1H), 10.62 (s, 1H), 9.76 (s, 1H),9.55 (s, 1H), 8.96 (t, J=5.9 Hz, 1H), 7.77 (dd, J=6.6, 2.6 Hz, 1H),7.54-7.44 (m, 2H), 7.42-7.35 (m, 2H), 7.34-7.26 (m, 2H), 6.58 (s, 1H),6.35 (s, 1H), 5.13 (dd, J=13.3, 5.1 Hz, 1H), 4.93-4.86 (m, 1H), 4.40 (q,J=17.6 Hz, 2H), 4.10 (q, J=5.3 Hz, 1H), 3.92 (s, 1H), 3.77 (s, 1H), 3.49(s, 2H), 3.30 (s, 2H), 3.20-3.13 (m, 5H), 2.96-2.83 (m, 4H), 2.67-2.60(m, 2H), 2.39-2.29 (m, 1H), 2.06-1.89 (m, 5H), 1.90 (s, 1H), 1.53-1.47(m, 1H), 1.04 (t, J=7.2 Hz, 3H), 0.81 (d, J=6.9 Hz, 6H); ESMS calculated(C₄₆H₅₃N₉O₉): 875.4; found: 876.4 (M+H).

SDC-TRAP-0124

(1-(1-(4-(3-(2,4-Dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzyl)piperidine-4-carbonyl)piperidin-4-yl)methyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

ESMS calculated (C₄₇H₅₅N₉O₉): 889.4; found: 890.3 (M+H).

SDC-TRAP-0125

(1-(4-(4-(3-(2,4-Dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenoxy)benzoyl)piperidin-4-yl)methyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 11.03 (s, 1H), 10.41 (s, 1H), 9.77 (s, 1H),9.55 (s, 1H), 8.99 (t, J=5.9 Hz, 1H), 7.77 (d, J=6.8 Hz, 1H), 7.54-7.42(m, 4H), 7.41-7.34 (m, 2H), 7.14-7.04 (m, 4H), 6.68 (s, 1H), 6.35 (s,1H), 5.13 (dd, J=13.3, 5.1 Hz, 1H), 4.39 (q, J=17.6 Hz, 2H), 4.03 (q,J=7.1 Hz, 2H), 3.19 (p, J=6.9 Hz, 2H), 3.03-2.85 (m, 2H), 2.60 (d,J=16.8 Hz, 1H), 2.36-2.29 (m, 1H), 1.99 (s, 3H), 1.75 (s, 2H), 1.29-1.13(m, 5H), 1.06 (t, J=7.2 Hz, 3H), 0.92 (d, J=6.9 Hz, 6H); ESMS calculated(C₄₇H₄₈N₈O₁₀): 884.3; found: 885.3 (M+H).

SDC-TRAP-0155

(1-((5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1-methyl-1H-indol-2-yl)methyl)piperidin-4-yl)methyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

ESMS calculated (C₄₁H₄₄N₈O₈): 776.3; found: 777.3 (M+H).

SDC-TRAP-0156

4-(4-(4-(3-(2,4-Dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenyl)piperazine-1-carbonyl)benzyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

ESMS calculated (C₄₃H₄₂N₈O₉): 814.3; found: 815.0 (M+H).

SDC-TRAP-0157

4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazine-1-carbonyl)benzyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

ESMS calculated (C₄₄H₄₃N₈O₉): 846.3; found: 847.2 (M+H).

SDC-TRAP-0160

5-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazine-1-carbonyl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)pyrazine-2-carboxamide

ESMS calculated (C₄₁H₃₉FN₁₀O₈): 818.3; found: 819.2 (M+H).

SDC-TRAP-0167

4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenoxy)phenyl(2-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamoyl)oxy)ethyl)(methyl)carbamate

ESMS calculated (C₄₄H₄₄N₈O₁₁): 860.3; found: 861.1 (M+H).

SDC-TRAP-0168

4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazine-1-carbonyl)-2,6-dimethylphenyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

ESMS calculated (C₄₅H₄₅FN₈O₉): 860.3; found: 861.2 (M+H).

SDC-TRAP-0170

5-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)pyrazine-2-carboxamide

To a solution of lenalidomide (0.2 g, 0.77 mmol) in DMF (4 mL) was added5-chloropyrazine-2-carboxylic acid (0.15 g, 0.95 mmol), HATU, (0.29 g,0.77 mmol), and DIPEA (0.27 mL, 1.54 mmol). The reaction was stirred atroom temperature for 1 hr before it was quenched with saturated NH₄Cl (5mL). The mixture was extracted with EtOAc (10 mL×3), and the combinedorganic phase was dried over Na₂SO₄ and concentrated. Columnchromatography gave5-chloro-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)pyrazine-2-carboxamide(0.1 g, 33%).

The solution of5-chloro-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)pyrazine-2-carboxamide (0.05 g, 0.13 mmol),4-(4-(3-fluoro-4-(piperazin-1-ylmethyl)phenyl)-5-hydroxy-4H-1,2,4-triazol-3-yl)-6-isopropylbenzene-1,3-diol(0.06 g, 0.13 mmol), and K₂CO₃ (0.07 g, 0.51 mmol) in DMF (3 mL) washeated in a microwave at 50° C. for 1 hr. The solution was diluted withsaturated NH₄Cl (5 mL), extracted with EtOAc (10 mL×3) and the combinedorganic phase was dried over Na₂SO₄ and concentrated. Columnchromatography gave SDC-TRAP-0170 (0.86 g, 87%).

¹H NMR (400 MHz, DMSO-d₆) δ 12.00 (s, 1H), 11.00 (s, 1H), 10.29 (s, 1H),9.64 (s, 1H), 9.41 (s, 1H), 8.73 (d, J=1.2 Hz, 1H), 8.34 (d, J=1.4 Hz,1H), 7.85 (dd, J=7.6, 1.4 Hz, 1H), 7.62-7.50 (m, 2H), 7.44 (t, J=8.2 Hz,1H), 7.09 (dd, J=10.8, 2.0 Hz, 1H), 6.99 (dd, J=8.2, 2.0 Hz, 1H), 6.87(s, 1H), 6.27 (s, 1H), 5.14 (dd, J=13.3, 5.1 Hz, 1H), 4.55-4.38 (m, 2H),3.74 (t, J=4.8 Hz, 4H), 3.59 (s, 2H), 3.33 (s, 2H), 3.17 (d, J=5.3 Hz,1H), 3.06-2.83 (m, 2H), 2.63-2.53 (m, 2H), 2.48-2.32 (m, 1H), 2.03-1.95(m, 1H), 1.00 (d, J=6.9 Hz, 6H); ESMS calculated (C₄₀H₃₉FN₁₀O₇): 790.3;found: 791.2 (M+H).

SDC-TRAP-0171

4-((((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamoyl)oxy)methyl)phenyl-4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazine-1-carboxylate

To a solution of 4-(hydroxymethyl)phenol (2 g, 16.1 mmol) in DMF (20 mL)was added TBSCl (2.7 g, 17.9 mmol) and imidazole (2.2 g, 32.3 mmol). Thereaction was stirred at room temperature for 2 hr. The reaction wasdiluted with EtOAc (100 mL) and washed with 0.1 N HCl (50 mL×3). Theorganic phase was dried over Na₂SO₄ and concentrated. Columnchromatography gave 4-(((tert-butyldimethylsilyl)oxy)methyl)phenol (2.6g, 68%).

To the solution of 4-(((tert-butyldimethylsilyl)oxy)methyl)phenol (1.0g, 4.2 mmol) in DCM (15 mL) was added 4-nitrophenyl chloroformate (1.0g, 4.96 mmol) followed by TEA (1.8 mL, 12.9 mmol). The reaction wasstirred at room temperature overnight. The reaction solution wasconcentrated and column chromatography gave4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl (4-nitrophenyl) carbonate(1.44 g, 85%).

To a solution of4-(4-(3-fluoro-4-(piperazin-1-ylmethyl)phenyl)-5-hydroxy-4H-1,2,4-triazol-3-yl)-6-isopropylbenzene-1,3-diol(0.32 g, 0.75 mmol) in DMF (5 mL) was added4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl (4-nitrophenyl) carbonate(0.36 g, 0.89 mmol) and TEA (0.31 mL, 2.22 mmol). The reaction wasstirred at room temperature for 1 hr before it was quenched withsaturated NH₄Cl (10 mL). The mixture was extracted with EtOAc (20 mL×2)and the combined organic phase was dried over Na₂SO₄ and concentrated.Column chromatography gave4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazine-1-carboxylate(0.38 g, 75%).

A solution of4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazine-1-carboxylate(0.38 g, 0.55 mmol) and TBAF (0.29 g, 1.10 mmol) was heated at 40° C.for 30 min. The solution was concentrated and column chromatography gave4-(hydroxymethyl)phenyl4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazine-1-carboxylate(0.22 g, 70%).

A solution of lenalidomide (1.0 g, 3.86 mmol) and 4-nitrophenylchloroformate (1.15 g, 5.70 mmol) was heated at 65° C. for 1 hr. Thesolution was allowed to cool to room temperature, then filtered. Thesolid was dried and used for the next step without further purification.

To the solution of 4-(hydroxymethyl)phenyl4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazine-1-carboxylate(0.23 g, 0.39 mmol) in DMF (4 mL) was added 4-nitrophenyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate (0.27 g,0.62 mmol) and TEA (0.17 mL, 1.17 mmol). The reaction was stirred atroom temperature overnight before it was quenched with NH₄Cl (5 mL). Themixture was extracted with EtOAc (20 mL×2) and combined organic phasewas dried over Na₂SO₄ and concentrated. Column chromatography gaveSDC-TRAP-0171 (0.21 g, 65%) as an off-white solid.

¹H NMR (400 MHz, DMSO-d₆) δ 11.96 (s, 1H), 10.98 (s, 1H), 9.65 (s, 1H),9.59 (s, 1H), 9.37 (s, 1H), 7.79 (dd, J=6.5, 2.5 Hz, 1H), 7.54-7.37 (m,5H), 7.18-7.04 (m, 3H), 6.99 (dd, J=8.1, 2.0 Hz, 1H), 6.87 (s, 1H), 6.27(s, 1H), 5.19-5.06 (m, 3H), 4.38 (q, J=17.6 Hz, 2H), 4.11-3.98 (m, 1H),3.57 (s, 3H), 3.41 (d, J=7.6 Hz, 1H), 3.28 (s, 1H), 3.17 (d, J=5.3 Hz,1H), 3.07-2.83 (m, 2H), 2.60 (d, J=17.3 Hz, 1H), 2.45 (s, 3H), 2.39-2.24(m, 1H), 2.04-1.99 (m, 1H), 1.00 (d, J=6.9 Hz, 6H); ESMS calculated(C₄₄H₄₃FN₈O₁₀): 862.3; found: 863.2 (M+H).

SDC-TRAP-0182

4-((((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamoyl)oxy)methyl)-2,6-dimethylphenyl4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazine-1-carboxylate

¹⁻H NMR (400 MHz, DMSO-d₆) δ 11.99 (s, 1H), 11.02 (s, 1H), 9.65 (d,J=13.1 Hz, 2H), 9.41 (s, 1H), 7.79 (dd, J=6.8, 2.3 Hz, 1H), 7.54-7.38(m, 3H), 7.16 (s, 2H), 7.08 (dd, J=11.0, 2.0 Hz, 1H), 6.99 (dd, J=8.2,2.0 Hz, 1H), 6.88 (s, 1H), 6.27 (s, 1H), 5.17-5.06 (m, 3H), 4.47-4.29(m, 2H), 3.72-3.61 (m, 2H), 3.56 (s, 2H), 3.44 (d, J=6.5 Hz, 2H),3.07-2.84 (m, 2H), 2.65-2.55 (m, 1H), 2.45 (s, 4H), 2.38-2.23 (m, 1H),2.10 (s, 6H), 2.05-1.96 (m, 1H), 1.01 (d, J=6.9 Hz, 6H); ESMS calculated(C₄₆H₄₇FN₈O₁₀): 890.3; found: 891.2 (M+H).

SDC-TRAP-0187

4-((((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamoyl)oxy)methyl)-2,6-dimethylphenyl4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenoxy)piperidine-1-carboxylate

ESMS calculated (C₄₉H₅₂N₈O₁₁): 928.4; found: 929.1 (M+H).

SDC-TRAP-0017

3-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)propanamide

ESMS calculated for C₃₅H₃₃N₇O₇: 663.24; Found: 664.2 (M+H)⁺.

SDC-TRAP-0015

N1-(2-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethoxy)ethyl)-N5-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)glutaramide

¹H NMR (400 MHz, DMSO-d₆) δ 11.87 (s, 1H), 11.02 (s, 1H), 9.90 (s, 1H),9.52 (s, 1H), 9.47 (s, 1H), 7.97-7.83 (m 2H), 7.55-7.38 (m, 4H), 6.92(d, J=8.7 Hz, 1H), 6.73 (s, 1H), 6.41 (s, 1H), 6.23 (s, 1H), 5.13 (d,J=13.6 Hz, 1H), 4.37 (dd, J=26.6, 17.5 Hz, 4H), 3.70-3.39 (m, 6H), 2.91(q, J=12.5, 11.7 Hz, 3H), 2.37 (d, J=8.9 Hz, 4H), 2.13 (t, J=7.3 Hz,2H), 2.06-1.96 (m, 2H), 1.86-1.77 (m, 2H), 1.22-0.90 (m, 2H), 0.83 (d,J=6.7 Hz, 6H). ESMS calculated for C₄₁H₄₄N₈O₉: 792.32; Found: 793.2(M+H)⁺.

SDC-TRAP-0018

N1-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)-N5-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)-N1-methylglutaramide

¹H NMR (400 MHz, DMSO-d₆) δ 11.94 (bs, 1H), 11.01 (s, 1H), 9.79 (s, 1H),9.45 (d, J=7.0 Hz, 2H), 7.79 (dd, J=18.5, 7.1 Hz, 1H), 7.50-7.38 (m,5H), 6.94 (t, J=7.6 Hz, 1H), 6.74 (d, J=9.7 Hz, 1H), 6.44 (s, 1H), 6.23(s, 1H), 5.14 (dd, J=12.6, 6.1 Hz, 1H), 4.49-4.24 (m, 4H), 3.65-3.54 (m,4H), 3.17 (d, J=4.6 Hz, 1H), 2.89 (d, J=12.7 Hz, 5H), 2.76 (s, 2H),2.45-2.24 (m, 4H), 2.13-1.97 (m, 4H), 1.80 (d, J=13.2 Hz, 2H), 1.60-1.52(m, 1H), 0.82 (d, J=7.9 Hz, 6H). ESMS calculated for C₄₀H₄₂N₈O₈: 762.31;Found: 763.2 (M+H)⁺.

SDC-TRAP-0021

2-(3-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)-3-methylureido)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)acetamide

¹H NMR (400 MHz, DMSO-d₆) δ 11.87 (s, 1H), 11.01 (s, 1H), 9.83 (s, 1H),9.53 (s, 1H), 9.47 (s, 1H), 7.86 (dd, J=6.3, 2.7 Hz, 1H), 7.58-7.46 (m,3H), 7.41 (dd, J=8.3, 2.6 Hz, 2H), 6.94 (dd, J=8.7, 2.0 Hz, 1H),6.82-6.70 (m, 2H), 6.43 (dd, J=3.2, 0.8 Hz, 1H), 6.23 (s, 1H), 5.14 (dd,J=13.3, 5.1 Hz, 1H), 4.46-4.26 (m, 4H), 3.91-3.84 (m, 2H), 3.59-3.50 (m,2H), 2.97-2.83 (m, 2H), 2.59 (s, 4H), 2.36-2.20 (m, 1H), 1.99 (s, 1H),0.82 (d, J=6.8 Hz, 6H). ESMS calculated for C₃₈H₃₉N₉O₈: 749.29; Found:750.2 (M+H)⁺.

SDC-TRAP-0033

N1-(2-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethoxy)ethyl)-N4-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)-N1-methylsuccinamide

¹H NMR (400 MHz, DMSO-d₆) δ 11.89 (m, 1H), 11.03 (s, 1H), 9.86 (s, 1H),9.58 (s, 1H), 9.50 (s, 1H), 7.94-7.81 (m, 2H), 7.74-7.30 (m, 7H), 6.93(d, J=8.7 Hz, 1H), 6.74 (s, 1H), 6.42 (d, J=7.5 Hz, 1H), 6.24 (s, 1H),5.15 (d, J=12.7 Hz, 1H), 4.51-4.37 (m, 4H), 3.86-3.42 (m, 5H), 3.19 (m,1H), 2.90-2.51 (m, 9H), 2.31-2.04 (m, 4H), 0.84 (d, J=5.9 Hz, 6H). ESMScalculated for C₄₁H₄₄N₈O₉: 792.32; Found: 793.3 (M+H)⁺.

SDC-TRAP-0041

5-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)-5-oxopentanamide

¹H NMR (400 MHz, DMSO-d₆) δ 11.94 (s, 1H), 11.03 (s, 1H), 9.80 (s, 1H),9.62 (s, 1H), 9.42 (s, 1H), 7.83 (dd, J=6.9, 2.1 Hz, 1H), 7.50 (d, J=7.1Hz, 2H), 7.31 (d, J=8.0 Hz, 2H), 7.15 (d, J=7.9 Hz, 2H), 6.78 (s, 1H),6.27 (s, 1H), 5.15 (dd, J=13.2, 5.1 Hz, 1H), 4.45-4.29 (m, 2H),3.62-3.54 (m, 1H), 3.44 (dd, J=14.8, 8.9 Hz, 8H), 3.03-2.85 (m, 2H),2.60 (dd, J=22.9, 8.3 Hz, 2H), 2.49-2.25 (m, 10H), 2.08-1.97 (m, 1H),1.82 (p, J=7.4 Hz, 2H), 0.95 (d, J=6.9 Hz, 6H). ESMS calculated forC₄₀H₄₄N₈O₈: 764.33; Found: 765.3 (M+H)⁺.

SDC-TRAP-0109

4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)piperazine-1-carboxamide

¹H NMR (400 MHz, DMSO-d₆) δ 11.94 (s, 1H), 10.99 (s, 1H), 9.61 (s, 1H),9.42 (s, 1H), 8.57 (s, 1H), 7.53-7.39 (m, 3H), 7.33 (d, J=8.0 Hz, 2H),7.15 (d, J=8.0 Hz, 2H), 6.77 (s, 1H), 6.27 (s, 1H), 5.12 (dd, J=13.2,5.2 Hz, 1H), 4.36-4.30 (m, 2H), 3.53-3.41 (m, 6H), 3.38 (s, 1H), 2.92(ddd, J=31.5, 15.9, 6.1 Hz, 2H), 2.64-2.54 (m, 1H), 2.47-2.35 (m, 5H),0.94 (d, J=6.9 Hz, 6H). ESMS calculated for C₃₆H₃₈N₈O₇: 694.29; Found:695.2 (M+H)⁺.

SDC-TRAP-0110

2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenyl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)acetamide

¹H NMR (400 MHz, DMSO-d₆) δ 11.83 (s, 1H), 11.00 (s, 1H), 9.77 (s, 1H),9.57 (s, 1H), 9.44 (s, 1H), 7.80 (dd, J=7.5, 1.5 Hz, 1H), 7.58-7.47 (m,2H), 7.06-6.98 (m, 2H), 6.97-6.89 (m, 2H), 6.78 (s, 1H), 6.27 (s, 1H),5.12 (dd, J=13.3, 5.1 Hz, 1H), 4.47-4.32 (m, 2H), 3.23 (d, J=5.8 Hz,6H), 3.03-2.83 (m, 3H), 2.76-2.55 (m, 6H), 2.47-2.32 (m, 1H), 2.02 (td,J=7.5, 3.9 Hz, 1H), 0.96 (d, J=6.9 Hz, 6H).

ESMS calculated for C₃₆H₃₈N₈O₇: 694.29; Found: 695.2 (M+H)⁺.

4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)piperidine-1-carboxamide

ESMS calculated for C₃₇H₃₉N₇O₇: 693.29; Found: 694.2 (M+H)⁺.

SDC-TRAP-0115

N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)-1-(4-(3-(2-hydroxy-5-isopropyl-4-methoxyphenyl)-5-(isopropylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzyl)piperidine-4-carboxamide

ESMS calculated for C₄₂H₄₈N₈O₇: 776.36; Found: 777.3 (M+H)⁺.

SDC-TRAP-0116

2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperidin-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)acetamide

¹H NMR (400 MHz, DMSO-d₆) δ 11.91 (s, 1H), 11.01 (s, 1H), 9.69 (s, 1H),9.58 (s, 1H), 9.42 (s, 1H), 7.77 (dd, J=7.5, 1.5 Hz, 1H), 7.58-7.46 (m,2H), 7.18 (d, J=8.4 Hz, 2H), 7.14-7.06 (m, 2H), 6.74 (s, 1H), 6.27 (s,1H), 5.13 (dd, J=13.2, 5.1 Hz, 1H), 4.45-4.30 (m, 2H), 3.20-3.09 (m,3H), 3.03-2.83 (m, 4H), 2.60 (ddd, J=17.4, 4.3, 2.4 Hz, 1H), 2.37 (qd,J=12.5, 11.8, 5.9 Hz, 1H), 2.14-1.96 (m, 3H), 1.60-1.44 (m, 3H),1.38-1.24 (m, 2H), 0.92 (d, J=6.9 Hz, 6H).

ESMS calculated for C₃₈H₄₁N₇O₇: 707.31; Found: 708.2 (M+H)⁺.

SDC-TRAP-0119

4-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)piperidine-1-carboxamide

¹H NMR (400 MHz, DMSO-d₆) δ 11.90 (s, 1H), 10.99 (s, 1H), 9.54 (d,J=17.1 Hz, 2H), 8.50 (s, 1H), 7.53-7.41 (m, 6H), 6.95 (d, J=8.7 Hz, 1H),6.69 (s, 1H), 6.47-6.41 (m, 1H), 6.25 (s, 1H), 5.12 (dd, J=13.1, 5.2 Hz,1H), 4.33 (s, 2H), 4.24 (t, J=6.9 Hz, 2H), 4.11-3.99 (m, 2H), 2.90 (td,J=13.9, 6.3 Hz, 2H), 2.75 (t, J=12.8 Hz, 2H), 2.60-2.55 (m, 1H),(2.45-2.34 (m, 1H), 2.00 (d, J=8.5 Hz, 1H), 1.74 (d, J=13.1 Hz, 4H),1.43 (s, 1H), 1.21-1.07 (m, 2H), 0.80 (d, J=6.8 Hz, 6H).

ESMS calculated for C₄₀H₄₂N₈O₇: 746.32; Found: 747.3 (M+H)⁺.

SDC-TRAP-0120

N1-(2-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethoxy)ethyl)-N5-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)-N1-methylglutaramide

¹H NMR (400 MHz, DMSO-d₆) δ 11.87 (s, 1H), 11.02 (s, 1H), 9.80 (d, J=4.4Hz, 1H), 9.54 (s, 1H), 9.47 (s, 1H), 7.82 (dt, J=7.4, 2.1 Hz, 1H),7.54-7.31 (m, 5H), 6.91 (dd, J=8.7, 2.0 Hz, 1H), 6.73 (d, J=2.1 Hz, 1H),6.40 (dd, J=7.0, 3.1 Hz, 1H), 6.22 (s, 1H), 5.19 5.09 (m, 1H), 4.45-4.26(m, 4H), 3.70-3.63 (m, 2H), 3.49-3.33 (m, 4H), 2.98-2.80 (m, 4H), 2.75(s, 1H), 2.60 (ddd, J=17.1, 4.3, 2.3 Hz, 1H), 2.35 (ddd, J=31.6, 15.2,7.4 Hz, 5H), 1.80 (p, J=7.4 Hz, 2H), 0.83 (dd, J=6.9, 2.1 Hz, 6H). ESMScalculated for C₄₂H₄₆N₈O₉: 806.34; Found: 807.3 (M+H)⁺.

SDC-TRAP-0121

2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)-2-oxoethyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 11.93 (s, 1H), 11.01 (s, 1H), 9.77 (s, 1H),9.60 (s, 1H), 9.40 (s, 1H), 7.77 (dt, J=7.0, 3.6 Hz, 1H), 7.56-7.46 (m,2H), 7.32 (d, J=8.0 Hz, 2H), 7.15 (d, J=7.8 Hz, 2H), 6.78 (s, 1H), 6.27(s, 1H), 5.12 (dd, J=13.3, 5.1 Hz, 1H), 4.85 (s, 2H), 4.45-4.35 (m, 2H),3.49 (s, 2H), 3.44 (s, 3H), 3.03-2.84 (m, 2H), 2.61 (d, J=17.6 Hz, 1H),2.42-2.26 (m, 6H), 2.07-1.99 (m, 1H), 0.95 (d, J=6.9 Hz, 6H). ESMScalculated for C₃₈H₄₀N₈O₉: 752.29; Found: 753.3 (M+H)⁺.

SDC-TRAP-0128

2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)acetamide

¹H NMR (400 MHz, DMSO-d₆) δ 11.92 (s, 1H), 11.01 (s, 1H), 9.71 (s, 1H),9.59 (s, 1H), 9.40 (s, 1H), 7.79 (dd, J=7.4, 1.5 Hz, 1H), 7.58-7.46 (m,2H), 7.30 (d, J=8.0 Hz, 2H), 7.13 (d, J=8.0 Hz, 2H), 6.77 (s, 1H), 6.26(s, 1H), 5.12 (dd, J=13.2, 5.1 Hz, 1H), 4.45-4.29 (m, 2H), 3.46 (s, 2H),3.16 (s, 2H), 3.02-2.84 (m, 2H), 2.65-2.50 (m, 5H), 2.47-2.32 (m, 5H),1.99 (m, 1H), 0.94 (d, J=6.9 Hz, 6H). ESMS calculated for C₃₇H₄₀N₈O₇:708.30; Found: 709.3 (M+H)⁺.

SDC-TRAP-0129

2-(4-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)piperidin-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)acetamide

¹H NMR (400 MHz, DMSO-d₆) δ 11.89 (s, 1H), 11.00 (s, 1H), 9.70 (s, 1H),9.54 (d, J=14.6 Hz, 2H), 7.77 (dd, J=7.4, 1.5 Hz, 1H), 7.58-7.40 (m,5H), 6.94 (dd, J=8.7, 2.1 Hz, 1H), 6.67 (s, 1H), 6.43 (d, J=3.1 Hz, 1H),6.24 (s, 1H), 5.12 (dd, J=13.2, 5.1 Hz, 1H), 4.45-4.29 (m, 2H), 4.22 (t,J=7.2 Hz, 2H), 3.12 (s, 2H), 2.87 (q, J=6.9 Hz, 4H), 2.59 (d, J=17.3 Hz,1H), 2.46-2.33 (m, 1H), 2.09-2.04 (m, 5H), 1.69 (d, J=6.9 Hz, 4H),1.36-1.25 (m, 2H), 0.78 (d, J=6.8 Hz, 6H). ESMS calculated forC₄₁H₄₄N₈O₇: 760.33; Found: 761.2 (M+H)⁺.

SDC-TRAP-0131

2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperidin-1-yl)-2-oxoethyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 11.91 (s, 1H), 11.01 (s, 1H), 9.75 (s, 1H),9.60 (s, 1H), 9.42 (s, 1H), 7.81-7.74 (m, 1H), 7.54-7.46 (m, 2H), 7.19(d, J=8.0 Hz, 2H), 7.10 (d, J=7.8 Hz, 2H), 6.75 (s, 1H), 6.27 (s, 1H),5.12 (dd, J=13.2, 5.2 Hz, 1H), 4.90-4.75 (m, 2H), 4.45 (d, J=17.6 Hz,1H), 4.40-4.24 (m, 2H), 3.69 (d, J=13.1 Hz, 1H), 3.02-2.84 (m, 3H), 2.61(d, J=17.6 Hz, 2H), 2.34 (td, J=14.4, 9.8 Hz, 1H), 2.08-1.96 (m, 2H),1.75 (s, 1H), 1.59 (t, J=12.0 Hz, 2H), 1.26-1.08 (m, 2H), 1.01 (s, 1H),0.94 (d, J=6.9 Hz, 6H). ESMS calculated for C₃₉H₄₁N₇O₉: 751.30; Found:752.2 (M+H)⁺.

SDC-TRAP-0149

1-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzyl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)piperidine-4-carboxamide

¹H NMR (400 MHz, DMSO-d₆) δ 11.03 (s, 1H), 10.61 (s, 1H), 9.76 (d, J=9.5Hz, 2H), 8.97 (t, J=5.9 Hz, 1H), 7.82 (dd, J=7.2, 1.9 Hz, 1H), 7.55-7.44(m, 2H), 7.40 (d, J=8.3 Hz, 2H), 7.35-7.27 (m, 2H), 6.59 (s, 1H), 6.35(s, 1H), 5.15 (dd, J=13.3, 5.1 Hz, 1H), 4.44-4.28 (m, 2H), 3.51 (s, 2H),3.31 (s, 1H), 3.23-3.11 (m, 2H), 2.92 (dq, J=13.4, 7.5, 6.4 Hz, 4H),2.61 (d, J=17.6 Hz, 1H), 2.39 (dtt, J=26.4, 13.3, 6.3 Hz, 2H), 2.01 (dd,J=12.9, 8.7 Hz, 3H), 1.81 (d, J=12.2 Hz, 2H), 1.70 (q, J=11.4 Hz, 2H),1.04 (t, J=7.1 Hz, 3H), 0.82 (d, J=6.9 Hz, 6H). ESMS calculated forC₄₀H₄₄N₈O₇: 748.33; Found: 749.3 (M+H)⁺.

SDC-TRAP-0152

4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzoyl)piperazin-1-yl)phenyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

ESMS calculated for C₄₅H₄₅N₉O₉: 855.33; Found: 856.2 (M+H)⁺.

SDC-TRAP-0168

4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazine-1-carbonyl)-2,6-dimethylphenyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

ESMS calculated for C₄₅H₄₅FN₈O₉: 860.33; Found: 861.2 (M+H)⁺.

SDC-TRAP-0173

4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzoyl)piperazin-1-yl)-2-methoxyphenyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 11.04 (s, 1H), 10.22 (s, 1H), 10.08 (s, 1H),9.75 (s, 1H), 9.03 (t, J=6.2 Hz, 1H), 7.80 (s, 1H), 7.50-7.41 (m, 6H),7.04 (d, J=8.5 Hz, 1H), 6.73 (d, J=11.0 Hz, 2H), 6.56-6.49 (m, 1H), 6.33(s, 1H), 5.15 (dd, J=13.3, 5.1 Hz, 1H), 4.44-4.28 (m, 2H), 3.79 (s, 3H),3.29-3.13 (m, 8H), 2.95-2.55 (m, 2H), 2.36 (d, J=14.6 Hz, 1H), 2.11-2.02(m, 1H), 1.06 (t, J=7.4 Hz, 3H), 0.91 (d, J=6.9 Hz, 6H). ESMS calculatedfor C₄₆H₄₇N₉O₁₀: 885.34; Found: 886.3 (M+H)⁺.

SDC-TRAP-0174

4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazine-1-carbonyl)-3-fluorobenzyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

ESMS calculated for C₄₄H₄₂FN₈O₉: 864.30; Found: 865.2 (M+H)⁺.

SDC-TRAP-0175

4-(4-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)pyridin-2-yl)piperazine-1-carbonyl)benzyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

ESMS calculated for C₄₂H₄₁N₉O₉: 815.30; Found: 816.1 (M+H)⁺.

SDC-TRAP-0176

4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzoyl)piperazin-1-yl)-2-methylphenyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 11.03 (s, 1H), 10.25 (s, 1H), 10.11 (s, 1H),9.75 (s, 1H), 9.02 (t, J=6.1 Hz, 1H), 7.81 (p, J=3.5 Hz, 1H), 7.58-7.46(m, 4H), 7.42 (d, J=7.9 Hz, 2H), 7.04 (d, J=8.7 Hz, 1H), 6.92 (d, J=2.7Hz, 1H), 6.84 (dd, J=8.8, 2.9 Hz, 1H), 6.72 (s, 1H), 6.34 (s, 1H), 5.14(dd, J=13.2, 5.1 Hz, 1H), 4.51 (d, J=17.7 Hz, 1H), 4.42 (d, J=17.7 Hz,1H), 3.78 (s, 2H), 3.50 (s, 2H), 3.18 (dt, J=20.9, 11.0 Hz, 6H), 2.94(dp, J=18.6, 6.2, 4.7 Hz, 2H), 2.53-2.47 (m, 2H), 2.46-2.30 (m, 1H),2.18 (s, 3H), 2.04 (dd, J=11.6, 5.9 Hz, 1H), 1.07 (t, J=7.2 Hz, 3H),0.91 (d, J=6.8 Hz, 6H). ESMS calculated for C₄₆H₄₇N₉O₉: 869.35; Found:870.1 (M+H)⁺.

SDC-TRAP-0177

4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzoyl)piperazine-1-carbonyl)benzyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 11.03 (s, 1H), 10.19 (s, 1H), 9.73 (s, 2H),9.02 (t, J=6.0 Hz, 1H), 7.84-7.77 (m, 1H), 7.50 (dq, J=11.4, 6.5 Hz,8H), 7.40 (d, J=6.8 Hz, 2H), 6.70 (s, 1H), 6.33-6.28 (m, 1H), 5.23 (s,2H), 5.13 (dd, J=13.2, 5.1 Hz, 1H), 4.40 (d, J=17.8 Hz, 2H), 3.68 (d,J=24.7 Hz, 4H), 3.22-3.12 (m, 2H), 2.93 (d, J=12.6 Hz, 2H), 2.65-2.55(m, 1H), 2.30-2.25 (m, 1H), 2.02 (dd, J=15.0, 7.1 Hz, 1H), 1.05 (t,J=7.1 Hz, 3H), 0.88 (d, J=7.5 Hz, 6H). ESMS calculated for C₄₇H₄₇N₉O₁₀:897.34; Found: 898.1 (M+H)⁺.

SDC-TRAP-0178

4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzoyl)piperazin-1-yl)-2,6-dimethylphenyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 11.02 (s, 1H), 10.22 (s, 1H), 10.17 (s, 1H),9.74 (s, 1H), 9.02 (t, J=5.9 Hz, 1H), 7.86-7.77 (m, 1H), 7.58-7.46 (m,4H), 7.45-7.37 (m, 2H), 6.73 (d, J=11.9 Hz, 3H), 6.33 (s, 1H), 5.13 (dd,J=13.2, 5.1 Hz, 1H), 4.50 (d, J=17.6 Hz, 1H), 4.41 (d, J=17.6 Hz, 1H),3.76 (s, 2H), 3.48 (s, 2H), 3.25-3.13 (m, 4H), 3.02-2.85 (m, 2H),2.66-2.57 (m, 1H), 2.45-2.31 (m, 1H), 2.14 (s, 6H), 2.04-2.02 (m, 1H),1.06 (t, J=7.2 Hz, 3H), 0.91 (d, J=6.9 Hz, 6H). ESMS calculated forC₄₇H₄₉N₉O₉: 883.37; Found: 884.1 (M+H)⁺.

SDC-TRAP-0194

4-(2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazin-1-yl)-2-oxoethyl)benzyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 12.04 (s, 1H), 11.06 (s, 1H), 9.70 (d, J=7.6Hz, 2H), 9.45 (s, 1H), 7.88-7.81 (m, 1H), 7.59-7.49 (m, 2H), 7.42 (d,J=8.2 Hz, 3H), 7.31-7.24 (m, 2H), 7.12 (dd, J=10.5, 2.1 Hz, 1H), 7.02(dd, J=8.1, 2.1 Hz, 1H), 6.92 (s, 1H), 6.33 (s, 1H), 5.22-5.12 (m, 3H),4.56-4.35 (m, 2H), 3.73 (d, J=15.5 Hz, 2H), 3.57-3.46 (m, 6H), 3.13-2.89(m, 2H), 2.71-2.61 (m, 1H), 2.37 (h, J=6.4, 5.4 Hz, 5H), 2.12-1.99 (m,1H), 1.05 (d, J=6.9 Hz, 6H). ESMS calculated for C₄₅H₄₅FN₈O₉: 860.33;Found: 861.2 (M+H)⁺.

SDC-TRAP-0195

4-(4-(1-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzyl)piperidine-4-carbonyl)piperazin-1-yl)-2,6-dimethylphenyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

ESMS calculated for C₅₃H₆₀N₁₀O₉: 980.45; Found: 981.3 (M+H)⁺.

SDC-TRAP-0196

4-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)isoindolin-2-yl)methyl)-2,6-dimethoxyphenyl(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 11.03 (s, 1H), 10.56 (s, 1H), 10.15 (s, 1H),9.77 (s, 1H), 8.99 (t, J=5.9 Hz, 1H), 7.82 (dd, J=5.7, 3.2 Hz, 1H), 7.52(q, J=4.1, 3.4 Hz, 2H), 7.36-7.24 (m, 2H), 7.17 (dd, J=7.9, 2.1 Hz, 1H),6.79 (s, 2H), 6.57 (s, 1H), 6.33 (s, 1H), 5.14 (dd, J=13.2, 5.2 Hz, 1H),4.49 (d, J=17.7 Hz, 1H), 4.40 (d, J=17.6 Hz, 1H), 3.90 (d, J=16.3 Hz,5H), 3.79 (s, 6H), 3.17 (p, J=7.0 Hz, 2H), 2.92 (tt, J=12.5, 6.2 Hz,2H), 2.62 (d, J=16.8 Hz, 1H), 2.42-2.31 (m, 1H), 2.10-2.01 (m, 1H), 1.05(t, J=7.1 Hz, 3H), 0.85 (d, J=6.9 Hz, 6H). ESMS calculated forC₄₅H₄₆N₈O₁₀: 858.33; Found: 859.2 (M+H)⁺.

in vitro activity was determined for these compounds using the HER2degradation assay set forth herein:

HER2 Degradation SDC-TRAP-# IC₅₀ (nM) SDC-TRAP-0015 2347 SDC-TRAP-0017 >10,000    SDC-TRAP-0018 8205  SDC-TRAP-0021 >5000 SDC-TRAP-0033 >5000  SDC-TRAP-0041 >10000   SDC-TRAP-0109 >10000  SDC-TRAP-0110 >10000   SDC-TRAP-0114 4,311   SDC-TRAP-0115 1890 SDC-TRAP-0116 967 SDC-TRAP-0105 >10000   SDC-TRAP-0119 >10,000   SDC-TRAP-0108 >10,000    SDC-TRAP-0122 >10000   SDC-TRAP-0121 3,000  SDC-TRAP-0128 6,909   SDC-TRAP-0129 4,519   SDC-TRAP-0126 8,636  SDC-TRAP-0132 >5000  SDC-TRAP-0127 8,086   SDC-TRAP-0131 >5,000  SDC-TRAP-0123 657 SDC-TRAP-0135 9667  SDC-TRAP-0133 >10000  SDC-TRAP-0136 >5000  SDC-TRAP-0140 >5000  SDC-TRAP-0149 1692 SDC-TRAP-0231 696 SDC-TRAP-0152 254 SDC-TRAP-0124 358 SDC-TRAP-0125 312SDC-TRAP-0156 3495  SDC-TRAP-0157 696 SDC-TRAP-0167 2861  SDC-TRAP-0168276 SDC-TRAP-0173 323 SDC-TRAP-0174 693 SDC-TRAP-0160 239 SDC-TRAP-0170296 SDC-TRAP-0171 199 SDC-TRAP-0162 >5,000   SDC-TRAP-0147 4329 SDC-TRAP-0175 2,629   SDC-TRAP-0178 170 91   SDC-TRAP-0176 178SDC-TRAP-0177 4,352   SDC-TRAP-0182 359 SDC-TRAP-0194 2,121  SDC-TRAP-0166 >5,000   SDC-TRAP-0188 3,950   SDC-TRAP-0189 1,091  SDC-TRAP-0195  49 SDC-TRAP-0163 885 SDC-TRAP-0164 493SDC-TRAP-0190 >5000  SDC-TRAP-0191 1,177   SDC-TRAP-0192 >5000 SDC-TRAP-0196  89 SDC-TRAP-0187  72 SDC-TRAP-0193 266 SDC-TRAP-01551190 

Hsp90^(α) Binding Assay Data

Binding No STA EC₅₀ (nM) 1 SDC-TRAP-0196 93.11 2 SDC-TRAP-0115 203.2 3SDC-TRAP-0116 158.8 4 SDC-TRAP-0127 102.2

Mouse Plasma Stability Data

% Remaining Compound ID (1 h, 10 μM) SDC-TRAP-0187  102% SDC-TRAP-019666.2% SDC-TRAP-0147 98.1% SDC-TRAP-0167 51.2% SDC-TRAP-0163 93.0%SDC-TRAP-0164 98.0% SDC-TRAP-0171 17.7% SDC-TRAP-0178 82.0%SDC-TRAP-0195 98.4% SDC-TRAP-0115 85.9% SDC-TRAP-0116 91.1%SDC-TRAP-0121 89.1% SDC-TRAP-0127 87.3% SDC-TRAP-0124  112%SDC-TRAP-0125 99.4% SDC-TRAP-0231 98.3% SDC-TRAP-0156 90.3%SDC-TRAP-0157 81.4%

Tissue Distribution Data for SDC-TRAP-0116

Tumor Conc. Plasma Conc. (μM) (nmol/g of tissue) Tumor/Plasma Ratio SDC-SDC- SDC- Analyte TRAP- TRAP- TRAP- Target 0116 Lenalidomide 0116Lenalidomide 0116 Lenalidomide Time (h) 0.083 693 — 0.560 17.7 — 0.08560.03 — 0.15 1 65.2 — 1.76 13.7 — 0.736 0.21 — 0.42 6 0.595 — 0.113 6.09— 0.120 10.2 — 1.07 24 0.0111 — BQL 2.78 — BQL 251 — — 48 0.0315 — BQL1.46 — BQL 4.65 — —

Tissue Distribution Data for SDC-TRAP-0171

Tumor Conc. Plasma Conc. (μM) (nmol/g of tissue) Tumor/Plasma Ratio SDC-SDC- SDC- SDC- SDC- SDC- Analyte TRAP- TRAP- TRAP- TRAP- TRAP- TRAP-Target 0171 0080 Lenalidomide 0171 0080 Lenalidomide 0171 0080Lenalidomide Time (h) 0.083 618 0.0312 3.23 0.083 618 0.0312 0.0164 3.800.613 1 32.2 0.258 2.03 1 32.2 0.258 0.249 0.636 1.06 6 1.21 0.153 0.2526 1.21 0.153 3.10 2.09 1.16 24 0.00162 0.0574 BQL 24 0.00162 0.0574 4076.91 — 48 BQL 0.0143 BQL 48 BQL 0.0143 — 26.8 —

Tissue Distribution Data for SDC-TRAP-0178

Tumor Conc. Plasma Conc. (μM) (nmol/g of tissue) Tumor/Plasma Ratio SDC-SDC- SDC- SDC- SDC- SDC- Analyte TRAP- TRAP- TRAP- TRAP- TRAP- TRAP-Target 0178 0183 Lenalidomide 0178 0183 Lenalidomide 0178 0183Lenalidomide Time (h) 0.083 918 N/A 1.39 16.4 0.320 0.623 0.0179 — 0.4491 217 N/A 0.963 12.8 0.316 0.629 0.0589 — 0.653 6 4.51 N/A 0.00447 7.170.418 0.0532 1.59 — 11.9 24 0.0280 N/A BQL 2.81 0.556 BQL 100 — — 480.241 N/A BQL 1.01 0.508 BQL — — —

Tissue Distribution Data for SDC-TRAP-0195

Tumor Conc. Plasma Conc. (μM) (nmol/g of tissue) Tumor/Plasma Ratio SDC-SDC- SDC- SDC- SDC- SDC- Analyte TRAP- TRAP- TRAP- TRAP- TRAP- TRAP-Target 0195 0197 Lenalidomide 0195 0197 Lenalidomide 0195 0197Lenalidomide Time (h) 0.083 1220 N/A 0.923 17.1 0.206 0.477 0.0140 —0.517 1 211 N/A 0.511 23.0 0.305 0.402 0.109 — 0.786 6 7.23 N/A 0.0031617.1 0.662 0.0458 2.36 — 14.51 24 2.03 N/A BQL 11.2 1.60 BQL 5.50 — — 48BQL N/A BQL 12.6 2.64 BQL — — —

Example 26: SDC-TRAPs Comprising Pemetrexed Fragment

Exemplary Synthesis of SDC-TRAPs:

To a solution of pemetrexed-fragment 2 (60 mg, 0.2 mmol) and amineSDC-TRAP-0004 (82 mg, 0.2 mmol) in anhydrous DMF (3 mL) was added EDC(60 mg, 0.3 mmol). The reaction mixture was stirred at room temperaturefor 18 h. The reaction mixture was then diluted with water (5 mL) andextracted with ethyl acetate (100 mL). The organic phase was dried withsodium sulfate, filtered and evaporated, followed by flashchromatography (hexane-ethyl acetate 1:1 and ethyl acetate-methanol98:2) to give SDC-TRAP-0019 (95 mg, 70%) as a white solid.

4-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl)-N-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)-N-methylbenzamide

¹H NMR (400 MHz, DMSO-d₆) δ: 11.86 (s, 1H); 10.61 (s, 1H); 10.14 (s,1H); 9.51 (s, 1H); 9.47 (s, 1H); 7.59-7.45 (m, 2H); 7.28-6.96 (m, 5H);6.72 (m, 2H); 6.47 (s, 1H); 6.32 (s, 1H); 6.24 (s, 1H); 6.00 (bs, 2H);4.46-4.28 (m, 2H); 3.75-3.49 (m, 2H); 2.96-2.80 (m, 5H); 2.61 (s, 3H);0.81 (d, J=6.9 Hz, 6H). ESMS calculated for C₃₇H₃₇N₉O₅: 687.29; Found:688.2 (M+H)⁺.

SDC-TRAP-0020

4-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl)-N-(2-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethoxy)ethyl)benzamide

¹H NMR (400 MHz, DMSO-d₆), δ (ppm): 11.86 (s, 1H); 10.61 (s, 1H); 10.14(s, 1H); 9.51 (s, 1H); 9.47 (s, 1H); 7.59-7.45 (m, 2H); 7.28-6.96 (m,5H); 6.72 (m, 2H); 6.47 (s, 1H); 6.32 (s, 1H); 6.24 (s, 1H); 6.01 (s,2H); 4.33 (d, J=6.5 Hz, 2H), 3.73 (d, J=6.3 Hz, 2H), 3.54-3.46 (m, 2H);3.00-2.82 (m, 7H), 0.81 (d, J=6.9 Hz, 6H); ESMS calculated forC₃₈H₃₉N₉O₆: 717.30; Found: 718.2 (M+H)⁺.

SDC-TRAP-0068

2-amino-5-(4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazine-1-carbonyl)phenethyl)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one

¹H NMR (400 MHz, DMSO-d₆) δ 11.92 (s, 1H), 10.62 (d, J=2.2 Hz, 1H),10.15 (s, 1H), 9.60 (s, 1H), 9.38 (s, 1H), 7.34-7.22 (m, 6H), 7.17-7.10(m, 2H), 6.79 (s, 1H), 6.33 (d, J=2.2 Hz, 1H), 6.26 (s, 1H), 6.00 (s,2H), 3.48 (s, 2H), 3.33 (s, 2H), 3.03-2.88 (m, 3H), 2.84 (dd, J=9.5, 5.7Hz, 2H), 2.37-2.34 (m, 4H), 0.95 (d, J=6.9 Hz, 6H); ESMS calculated forC₃₇H₃₉N₉O₅: 689.31; Found: 690.1 (M+H)⁺.

SDC-TRAP-0078

2-amino-5-(4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazine-1-carbonyl)phenethyl)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one

¹H NMR (400 MHz, DMSO-d₆) δ 11.97 (s, 1H), 10.63 (d, J=2.3 Hz, 1H),10.15 (s, 1H), 9.63 (s, 1H), 9.39 (s, 1H), 7.96 (s, 1H), 7.40 (t, J=8.1Hz, 1H), 7.27 (s, 4H), 7.06 (dd, J=10.9, 2.1 Hz, 1H), 6.97 (dd, J=8.2,2.0 Hz, 1H), 6.88 (s, 1H), 6.34 (d, J=2.2 Hz, 1H), 6.26 (s, 1H), 6.00(s, 2H), 3.54 (bs, 4H), 3.07-2.80 (m, 3H), 2.74 (s, 2H), 2.40 (bs, 4H),1.01 (d, J=6.9 Hz, 6H). ESMS calculated for C₃₇H₃₈FN₉O₅: 707.30; Found:708.2 (M+H)⁺.

SDC-TRAP-0082

2-amino-5-(4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenyl)piperazine-1-carbonyl)phenethyl)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one

¹H NMR (400 MHz, DMSO-d₆) δ 11.85 (s, 1H), 10.63 (d, J=2.1 Hz, 1H),10.15 (s, 1H), 9.59 (s, 1H), 9.44 (s, 1H), 7.37-7.25 (m, 4H), 7.04 (d,J=8.6 Hz, 2H), 6.97-6.90 (m, 2H), 6.81 (s, 1H), 6.35 (d, J=2.2 Hz, 1H),6.27 (s, 1H), 6.01 (s, 2H), 3.69 (s, 2H), 3.52 (s, 2H), 3.18 (s, 4H),3.04-2.90 (m, 3H), 2.86 (dd, J=9.5, 5.8 Hz, 2H), 0.98 (d, J=6.9 Hz, 6H);ESMS calculated for C₃₆H₃₇N₉O₅: 675.29; Found: 676.2 (M+H)⁺.

SDC-TRAP-0093

2-amino-5-(4-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)isoindoline-2-carbonyl)phenethyl)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one

¹H NMR (400 MHz, DMSO-d₆) δ 11.91 (s, 1H), 10.64 (s, 1H), 10.23 (s, 1H),9.62 (s, 1H), 9.38 (s, 1H), 7.51 (dd, J=8.2, 3.4 Hz, 2H), 7.40-7.17 (m,4H), 7.07-6.96 (m, 1H), 6.91 (s, 1H), 6.36 (s, 1H), 6.25 (s, 1H), 6.06(s, 2H), 4.78 (dd, J=31.3, 14.1 Hz, 4H), 3.07-2.93 (m, 3H), 2.87 (dd,J=9.5, 5.8 Hz, 2H), 1.02 (dd, J=10.8, 6.8 Hz, 6H); ESMS calculated forC₃₄H₃₂N₈O₅: 632.25; Found: 633.1 (M+H)⁺.

SDC-TRAP-0102

2-amino-5-(4-(4-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1-methyl-1H-benzo[d]imidazol-2-yl)piperidine-1-carbonyl)phenethyl)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one

¹H NMR (400 MHz, DMSO-d₆) δ 11.86 (s, 1H), 10.66-10.60 (m, 1H), 10.17(s, 1H), 9.57 (s, 1H), 9.36 (s, 1H), 7.48 (d, J=8.7 Hz, 1H), 7.40-7.25(m, 4H), 7.06-6.99 (m, 1H), 6.86 (s, 1H), 6.35 (d, J=2.3 Hz, 1H), 6.20(s, 1H), 6.02 (s, 2H), 4.53 (s, 1H), 3.79 (s, 3H), 3.02-2.81 (m, 5H),1.95 (s, 2H), 1.76 (q, J=11.9 Hz, 2H), 0.96 (d, J=6.7 Hz, 6H); ESMScalculated for C₃₉H₄₀N₁₀O₅: 728.32; Found: 729.2 (M+H)⁺.

SDC-TRAP-0103

2-amino-5-(4-(4-((4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperidin-1-yl)methyl)piperidine-1-carbonyl)phenethyl)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one

¹H NMR (400 MHz, DMSO-d₆) δ 11.93 (s, 1H), 10.63 (s, 1H), 10.20 (s, 1H),9.69 (s, 1H), 9.49 (s, 1H), 7.20 (d, J=39.7 Hz, 6H), 7.08 (d, J=8.0 Hz,2H), 6.73 (s, 1H), 6.31 (d, J=19.5 Hz, 2H), 6.04 (s, 2H), 4.42 (s, 1H),3.58 (s, 1H), 2.95 (dt, J=13.8, 7.4 Hz, 4H), 2.85 (d, J=8.1 Hz, 2H),2.77 (d, J=10.7 Hz, 3H), 2.08 (d, J=6.7 Hz, 2H), 1.76-1.59 (m, 6H),1.51-1.43 (m, 3H), 1.12-0.89 (m, 6H); ESMS calculated for C₄₄H₅₁N₉O₅:785.40; Found: 786.3 (M+H)⁺.

SDC-TRAP-0130

2-amino-5-(4-(4-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)piperidine-1-carbonyl)phenethyl)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one

¹H NMR (400 MHz, DMSO-d₆) δ 11.88 (s, 1H), 10.62 (s, 1H), 10.17-10.11(m, 1H), 9.53 (dd, J=20.0, 2.8 Hz, 2H), 7.52-7.39 (m, 3H), 7.25 (d,J=2.8 Hz, 4H), 6.97-6.89 (m, 1H), 6.68 (d, J=2.7 Hz, 1H), 6.42 (t, J=3.1Hz, 1H), 6.33 (d, J=2.8 Hz, 1H), 6.23 (d, J=2.8 Hz, 1H), 6.00 (s, 2H),4.41 (s, 1H), 4.21 (t, J=7.4 Hz, 2H), 2.98-2.80 (m, 6H), 1.76-1.66 (m,4H), 1.47 (bs, 2H), 1.20-1.10 (m, 3H), 0.78 (dd, J=7.1, 2.7 Hz, 6H);ESMS calculated for C₄₁H₄₃N₉O₅: 741.34; Found: 742.3 (M+H)⁺.

in vitro activity was determined for these compounds using the HER2degradation assay set forth herein:

HER2 degradation SDC-TRAP-# IC₅₀ (nM) SDC-TRAP-0020 >5000 SDC-TRAP-00194419 SDC-TRAP-0068 262 SDC-TRAP-0078 1005 SDC-TRAP-0082 1042SDC-TRAP-0093 >5,000 SDC-TRAP-0102 >5,000 SDC-TRAP-0103 245SDC-TRAP-0130 1829

Mouse Plasma Stability

SDC-TRAP-# % Remaining (1 h) SDC-TRAP-0068 96.5% SDC-TRAP-0141  101%

Example 27: SDC-TRAPs Comprising SN-38

SDC-TRAP-0011

(S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenoxy)piperidine-1-carboxylate

¹H NMR (400 MHz, DMSO-d₆) δ 10.02 (s, 3H), 8.17 (d, J=9.2 Hz, 1H),8.01-7.93 (m, 1H), 7.74-7.62 (m, 2H), 7.18-7.01 (m, 4H), 6.70 (s, 1H),6.40 (s, 1H), 6.05 (s, 1H), 5.44 (d, J=4.7 Hz, 1H), 5.25 (s, 2H), 4.92(dd, J=11.8, 6.8 Hz, 1H), 4.69 (d, J=10.6 Hz, 2H), 4.03 (q, J=7.1 Hz,1H), 3.79 (s, 1H), 3.59 (s, 1H), 3.17 (q, J=7.6 Hz, 2H), 3.03-2.87 (m,2H), 2.55 (s, 1H), 2.21-1.96 (m, 2H), 1.73 (s, 2H), 1.30 (t, J=7.6 Hz,3H), 1.01-0.81 (m, 9H) ppm; ESMS calculated for C₄₅H₄₄N₆O₁₀: 828.3;found: 829.1 (M+H⁺).

SDC-TRAP-0012

(S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl-4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenoxy)piperidine-1-carboxylatehydrochloride

¹H NMR (400 MHz, DMSO-d₆) δ 11.88 (s, 1H), 10.34 (s, 1H), 9.60 (s, 1H),9.43 (s, 1H), 8.02 (t, J=10.0 Hz, 1H), 7.46-7.38 (m, 2H), 7.15-7.07 (m,2H), 6.98 (d, J=15.2 Hz, 3H), 6.78 (s, 1H), 6.27 (s, 1H), 5.45 (d, J=3.6Hz, 2H), 5.30 (d, J=2.4 Hz, 2H), 4.64 (d, J=9.6 Hz, 1H), 4.03 (m, 1H),3.57 (s, 1H), 3.20 (s, 1H), 3.09 (q, J=7.6 Hz, 3H), 2.98 (q, J=6.9 Hz,1H), 2.55 (s, 4H), 2.14 (q, J=11.2, 9.3 Hz, 3H), 1.46 (s, 1H), 1.29 (t,J=7.6 Hz, 3H), 0.99-0.87 (m, 9H). ppm; ESMS calculated for C₄₅H₄₄N₆O₁₀:828.3; found: 829.0 (M+H⁺).

SDC-TRAP-0014

(S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-((4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenoxy)methyl)piperidine-1-carboxylate

¹H NMR (400 MHz, Methanol-d₄) δ 8.07 (d, J=9.1 Hz, 1H), 7.91 (d, J=9.1Hz, 1H), 7.52-7.36 (m, 4H), 7.35-7.16 (m, 2H), 7.04 (d, J=8.4 Hz, 1H),6.94 (d, J=8.5 Hz, 1H), 6.57-6.49 (m, 1H), 6.37 (s, 1H), 5.67 (d, J=16.9Hz, 1H), 5.42 (d, J=17.0 Hz, 1H), 4.45 (s, 2H), 4.12-4.00 (m, 1H), 3.88(dd, J=17.8, 7.5 Hz, 1H), 3.78 (d, J=7.6 Hz, 1H), 3.39 (s, 2H), 3.14 (q,J=10.3, 6.7 Hz, 2H), 2.99 (dt, J=14.4, 7.1 Hz, 1H), 2.83 (d, J=14.9 Hz,1H), 2.37-1.96 (m, 5H), 1.86 (d, J=13.2 Hz, 2H), 1.77 (d, J=13.5 Hz,1H), 1.62 (td, J=27.9, 24.2, 13.8 Hz, 1H), 1.39 (t, J=7.6 Hz, 3H), 1.04(t, J=7.5 Hz, 3H), 0.91-0.73 (m, 6H). ppm; ESMS calculated forC₄₆H₄₆N₆O₁₀: 842.3; found: 843.1 (M+H⁺).

SDC-TRAP-0063

(S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)piperidine-1-carboxylate

¹H NMR (400 MHz, Chloroform-d) δ 8.21 (d, J=9.2 Hz, 1H), 7.84 (d, J=2.5Hz, 1H), 7.68 (s, 1H), 7.64-7.56 (m, 2H), 7.47 (d, J=8.7 Hz, 1H),7.24-7.12 (m, 2H), 6.55 (dd, J=3.2, 0.8 Hz, 1H), 6.37 (d, J=4.2 Hz, 2H),5.73 (d, J=16.3 Hz, 1H), 5.36-5.24 (m, 3H), 4.41 (d, J=13.5 Hz, 1H),4.29 (q, J=9.3, 7.5 Hz, 3H), 3.17 (q, J=7.7 Hz, 2H), 3.06 (t, J=12.7 Hz,1H), 2.96-2.77 (m, 2H), 2.42 (s, 2H), 1.90 (dq, J=14.2, 7.1 Hz, 6H),1.45-1.33 (m, 5H), 1.31-1.22 (m, 1H), 1.04 (t, J=7.3 Hz, 3H), 0.50 (d,J=6.8 Hz, 6H). ppm; ESMS calculated for C₄₉H₄₉N₇O₉: 879.4; found: 880.2(M+H⁺).

SDC-TRAP-0064

(S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)piperidine-1-carboxylate

ESMS calculated for C₄₉H₄₉N₇O₉: 879.4; found: 880.1 (M+H⁺).

SDC-TRAP-0065

(S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl(3-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)propyl)(methyl)carbamate

¹H NMR (400 MHz, Chloroform-d) δ 8.22 (dd, J=9.3, 2.0 Hz, 1H), 7.86 (dd,J=8.9, 2.6 Hz, 1H), 7.70 (d, J=2.2 Hz, 1H), 7.66-7.56 (m, 1H), 7.49 (d,J=7.9 Hz, 2H), 7.37-7.24 (m, 4H), 6.47 (d, J=16.0 Hz, 1H), 6.41-6.35 (m,1H), 5.72 (dd, J=16.2, 2.2 Hz, 1H), 5.37-5.26 (m, 3H), 4.0 (m, 1H), 3.57(d, J=4.1 Hz, 3H), 3.51-3.35 (m, 3H), 3.19 (d, J=8.4 Hz, 4H), 3.09 (d,J=2.2 Hz, 1H), 2.92 (dt, J=19.0, 7.0 Hz, 1H), 2.58-2.42 (m, 6H), 1.92(dq, J=15.4, 7.4 Hz, 5H), 1.41 (tt, J=7.7, 4.1 Hz, 4H), 1.32-1.22 (m,2H), 1.04 (t, J=7.4 Hz, 3H), 0.78-0.65 (m, 6H). ppm; ESMS calculated forC₄₉H₅₄N₈O₉: 898.4; found: 899.2 (M+H⁺).

SDC-TRAP-0066

(S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl(2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)ethyl)(methyl)carbamate

¹H NMR (400 MHz, Chloroform-d) δ 8.22 (dd, J=9.2, 2.9 Hz, 1H), 7.87 (t,J=2.5 Hz, 1H), 7.70 (d, J=1.3 Hz, 1H), 7.62 (ddd, J=8.7, 5.9, 2.4 Hz,1H), 7.51-7.44 (m, 2H), 7.31-7.23 (m, 2H), 6.47 (d, J=15.7 Hz, 1H),6.39-6.31 (m, 1H), 5.70 (d, J=16.4 Hz, 1H), 5.37-5.26 (m, 3H), 3.61-3.53(m, 3H), 3.43-3.33 (m, 3H), 3.25-3.13 (m, 3H), 3.10 (s, 1H), 2.96-2.84(m, 1H), 2.77-2.60 (m, 5H), 2.55 (s, 4H), 1.99-1.85 (m, 2H), 1.41 (t,J=7.7 Hz, 3H), 1.03 (t, J=7.3 Hz, 3H), 0.77-0.65 (m, 6H). ppm; ESMScalculated for C₄₈H₅₂N₈O₉: 884.4; found: 885.1 (M+H⁺).

SDC-TRAP-0084

(S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl(3-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)propyl)(methyl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 12.05 (s, 1H), 9.74 (s, 1H), 8.02 (dd,J=9.9, 6.7 Hz, 1H), 7.50 (t, J=7.7 Hz, 1H), 7.45-7.33 (m, 3H), 7.27-7.17(m, 2H), 7.01 (d, J=5.8 Hz, 1H), 6.85 (d, J=2.3 Hz, 1H), 6.26 (d, J=3.2Hz, 1H), 5.44 (d, J=2.4 Hz, 2H), 5.28 (s, 2H), 4.12 (d, J=16.9 Hz, 1H),3.96 (s, 1H), 3.69 (s, 2H), 3.64 (s, 1H), 3.31-3.22 (m, 1H), 3.18 (m,7H), 3.09 (d, J=16.2 Hz, 3H), 2.98 (p, J=6.8 Hz, 1H), 2.89 (s, 2H), 2.76(s, 1H), 2.46 (s, 2H), 2.20-2.05 (m, 2H), 1.84 (t, J=8.2 Hz, 1H), 1.27(td, J=7.7, 4.8 Hz, 3H), 1.02-0.85 (m, 9H). ppm; ESMS calculated forC₄₉H₅₄N₈O₉: 898.4; found: 899.3 (M+H⁺).

SDC-TRAP-0086

(S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenethyl)piperidine-1-carboxylate

¹H NMR (400 MHz, Chloroform-d) δ 8.21 (d, J=9.2 Hz, 1H), 7.85 (d, J=2.5Hz, 1H), 7.69-7.57 (m, 2H), 7.37 (d, J=7.9 Hz, 2H), 7.28 (d, J=8.8 Hz,2H), 6.44 (d, J=1.6 Hz, 1H), 6.37 (d, J=1.1 Hz, 1H), 5.74 (dt, J=16.3,1.2 Hz, 1H), 5.36-5.24 (m, 3H), 4.42 (d, J=13.4 Hz, 1H), 4.31 (d, J=13.3Hz, 1H), 3.23-3.03 (m, 3H), 2.94 (dq, J=14.0, 7.3 Hz, 2H), 2.76 (t,J=7.7 Hz, 2H), 2.05 (d, J=0.9 Hz, 1H), 1.91 (dq, J=14.6, 7.4 Hz, 4H),1.66 (d, J=7.7 Hz, 2H), 1.40 (q, J=9.8, 8.7 Hz, 5H), 1.08-0.89 (m, 3H),0.74 (d, J=6.8 Hz, 6H). ppm; ESMS calculated for C₄₇H₄₈N₆O₉: 840.4;found: 841.2 (M+H⁺).

SDC-TRAP-0088

(S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-((4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)methyl)piperidine-1-carboxylate

ESMS calculated for C₅₁H₅₆N₈O₉: 924.4; found: 925.4 (M+H⁺).

SDC-TRAP-0087

(S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl(2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)ethyl)(methyl)carbamate

¹H NMR (400 MHz, Methanol-d₄) δ 8.54 (s, 1H), 8.20 (s, 1H), 7.90-7.50(m, 4H), 7.41 (s, 1H), 7.28 (s, 1H), 6.90-6.20 (m, 2H), 5.70-5.30 (m,6H), 4.40-4.10 (m, 7H), 3.98 (s, 2H), 3.77 (s, 2H), 3.71 (s, 2H), 3.59(s, 2H), 3.37 (d, J=19.0 Hz, 5H), 3.05 (s, 1H), 2.94 (s, 1H), 1.44 (s,2H), 1.05 (dd, J=19.6, 6.6 Hz, 6H), 0.96 (d, J=6.6 Hz, 6H). ppm; ESMScalculated for C₄₈H₅₂N₈O₉: 884.4; found: 885.3 (M+H⁺).

SDC-TRAP-0089

(S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)isoindolin-2-yl)piperidine-1-carboxylate

¹H NMR (400 MHz, Chloroform-d) δ 8.22 (d, J=9.2 Hz, 1H), 7.87 (d, J=2.5Hz, 1H), 7.69 (s, 1H), 7.62 (dd, J=9.2, 2.5 Hz, 1H), 7.39 (d, J=7.8 Hz,1H), 7.20 (d, J=7.5 Hz, 2H), 6.49 (s, 1H), 6.36 (s, 1H), 5.71 (d, J=16.4Hz, 1H), 5.36-5.25 (m, 3H), 4.31 (d, J=13.3 Hz, 1H), 4.18 (d, J=13.3 Hz,1H), 4.11-4.03 (m, 4H), 3.42-3.30 (m, 1H), 3.19 (q, J=7.7 Hz, 1H), 3.00(h, J=7.4, 6.9 Hz, 1H), 2.81-2.71 (m, 1H), 2.09-2.00 (m, 2H), 1.98-1.85(m, 5H), 1.42 (t, J=7.7 Hz, 3H), 1.32-1.23 (m, 3H), 1.04 (t, J=7.4 Hz,3H), 0.79 (d, J=6.8 Hz, 6H). ppm; ESMS calculated for C₄₇H₄₇N₇O₉: 853.3;found: 854.3 (M+H⁺).

SDC-TRAP-0090

(S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)pyridin-2-yl)piperazine-1-carboxylate

¹H NMR (400 MHz, Chloroform-d) δ 8.25 (d, J=9.3 Hz, 1H), 8.12 (d, J=2.8Hz, 1H), 7.91 (d, J=2.7 Hz, 1H), 7.78-7.57 (m, 2H), 7.51 (dd, J=9.1, 2.8Hz, 1H), 6.85 (dd, J=9.4, 2.8 Hz, 1H), 6.62 (d, J=2.8 Hz, 1H), 6.39 (d,J=2.8 Hz, 1H), 5.71 (d, J=16.5 Hz, 1H), 5.39-5.22 (m, 4H), 4.07 (s, 1H),3.98-3.68 (m, 4H), 3.21 (d, J=7.8 Hz, 2H), 3.12-2.95 (m, 1H), 2.06 (d,J=2.8 Hz, 2H), 2.01-1.86 (m, 2H), 1.61 (d, J=7.0 Hz, 1H), 1.44 (td,J=7.7, 2.8 Hz, 4H), 1.26 (d, J=3.4 Hz, 2H), 1.05 (td, J=7.3, 2.8 Hz,3H), 0.94-0.80 (m, 6H). ppm; ESMS calculated for C₄₃H₄₂N₈O₉: 814.3;found: 815.2 (M+H⁺).

SDC-TRAP-0091

(S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)pyridin-2-yl)piperazine-1-carboxylate

¹H NMR (400 MHz, DMSO-d₆) δ 11.93 (s, 1H), 9.64 (s, 1H), 9.48 (s, 1H),7.99-7.87 (m, 2H), 7.49-7.37 (m, 3H), 7.04 (s, 1H), 6.98-6.91 (m, 2H),6.28 (s, 1H), 5.53-5.38 (m, 2H), 5.29 (d, J=1.8 Hz, 2H), 3.78-3.60 (m,4H), 3.51-3.34 (m, 4H), 3.14-2.95 (m, 3H), 2.14 (dd, J=14.3, 7.0 Hz,2H), 1.38-1.21 (m, 3H), 1.04 (dd, J=6.9, 1.9 Hz, 6H), 0.92 (t, J=7.4 Hz,3H). ppm; ESMS calculated for C₄₃H₄₂N₈O₉: 814.3; found: 815.2 (M+H⁺).

SDC-TRAP-0092

(S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)isoindolin-2-yl)piperidine-1-carboxylate

¹H NMR (400 MHz, Chloroform-d) δ 8.02 (d, J=9.1 Hz, 1H), 7.89 (d, J=9.1Hz, 1H), 7.47-7.37 (m, 1H), 7.30-7.20 (m, 2H), 7.17 (dd, J=9.8, 2.6 Hz,2H), 7.04 (s, 1H), 6.50 (d, J=27.1 Hz, 1H), 6.32 (d, J=4.2 Hz, 1H), 5.68(d, J=16.9 Hz, 1H), 5.40 (d, J=16.9 Hz, 1H), 5.18-4.87 (m, 2H),4.41-4.19 (m, 1H), 4.10-3.81 (m, 4H), 3.76-3.60 (m, 1H), 3.48-3.36 (m,1H), 3.09-2.85 (m, 6H), 2.72 (s, 1H), 2.28 (dd, J=13.8, 7.5 Hz, 1H),2.22-2.08 (m, 1H), 1.88 (d, J=10.1 Hz, 1H), 1.68-1.54 (m, 1H), 1.35-1.18(m, 3H), 1.02 (dt, J=12.6, 6.1 Hz, 3H), 0.85-0.69 (m, 6H). ppm; ESMScalculated for C₄₇H₄₇N₇O₉: 853.3; found: 854.2 (M+H⁺).

SDC-TRAP-0104

(S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenethyl)piperidine-1-carboxylate

¹H NMR (400 MHz, Chloroform-d) δ 8.44 (d, J=9.2 Hz, 1H), 8.11-7.96 (m,2H), 7.72 (s, 1H), 7.53 (d, J=9.2 Hz, 1H), 7.35 (s, 1H), 7.30-7.13 (m,4H), 6.50-6.29 (m, 2H), 5.68 (d, J=17.3 Hz, 1H), 5.40 (d, J=17.3 Hz,1H), 5.18 (t, J=5.4 Hz, 2H), 4.42 (dd, J=24.8, 13.2 Hz, 1H), 4.05-3.89(m, 1H), 3.44 (s, 3H), 2.84-2.60 (m, 4H), 2.44-2.10 (m, 2H), 1.94-1.80(m, 5H), 1.61 (dd, J=11.7, 3.7 Hz, 3H), 1.36 (dt, J=12.3, 4.9 Hz, 3H),1.05 (dq, J=13.8, 7.0 Hz, 3H), 0.78-0.61 (m, 6H). ppm; ESMS calculatedfor C₄₇H₄₈N₆O₉: 840.4; found: 841.2 (M+H⁺).

SDC-TRAP-0106

(S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenethyl)piperidin-1-yl)acetate

¹H NMR (400 MHz, Chloroform-d) δ 8.00 (d, J=9.1 Hz, 1H), 7.39 (dd,J=5.2, 2.5 Hz, 1H), 7.31 (d, J=2.6 Hz, 1H), 7.29-7.14 (m, 4H), 6.40 (d,J=23.7 Hz, 2H), 5.68 (d, J=17.0 Hz, 1H), 5.42 (dd, J=17.0, 3.1 Hz, 1H),5.22 (s, 2H), 3.11 (q, J=7.9 Hz, 2H), 2.98-2.81 (m, 2H), 2.59 (dt,J=10.3, 4.7 Hz, 2H), 2.45-2.08 (m, 6H), 1.80-1.44 (m, 4H), 1.44-1.19 (m,6H), 0.99 (t, J=7.4 Hz, 3H), 0.70 (dd, J=6.8, 2.3 Hz, 6H). ppm; ESMScalculated for C₄₈H₅₀N₆O₉: 854.4; found: 855.3 (M+H⁺).

SDC-TRAP-0107

(S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl2-(4-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)piperidin-1-yl)acetate

¹H NMR (400 MHz, Chloroform-d) δ 8.07-7.92 (m, 1H), 7.54 (d, J=7.2 Hz,1H), 7.36 (dq, J=5.9, 3.7 Hz, 5H), 7.30-7.19 (m, 1H), 7.19-6.99 (m, 2H),6.47 (d, J=3.5 Hz, 1H), 6.41-6.27 (m, 2H), 5.75-5.59 (m, 1H), 5.41 (d,J=17.1 Hz, 1H), 5.21 (s, 2H), 4.26-3.94 (m, 2H), 3.51-3.24 (m, 5H), 3.11(q, J=7.6 Hz, 2H), 2.93 (t, J=13.0 Hz, 2H), 2.80 (q, J=6.8 Hz, 1H), 2.23(ddd, J=36.9, 13.1, 7.3 Hz, 4H), 1.71 (td, J=14.1, 13.5, 5.4 Hz, 4H),1.48-1.15 (m, 5H), 1.05-0.89 (m, 3H), 0.52-0.32 (m, 6H). ppm; ESMScalculated for C₅₀H₅₁N₇O₉: 893.4; found: 894.3 (M+H⁺).

SDC-TRAP-0145

(S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenoxy)phenyl)(methyl)carbamate

ESMS calculated for C₅₀H₄₇N₇O₁₀: 905.3; found: 906.3 (M+H⁺).

SDC-TRAP-0204

(S)—(S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenyl)piperazine-1-carbonyl)pyrrolidine-1-carboxylate

¹H NMR (400 MHz, Chloroform-d) δ 8.20 (dd, J=9.2, 5.6 Hz, 1H), 7.86 (dd,J=42.0, 2.5 Hz, 1H), 7.72-7.50 (m, 2H), 7.22-7.08 (m, 2H), 6.95 (dd,J=35.5, 8.8 Hz, 2H), 6.49-6.25 (m, 2H), 5.72 (dd, J=16.4, 4.4 Hz, 1H),5.42-5.23 (m, 3H), 5.05-4.79 (m, 1H), 4.05-3.51 (m, 5H), 3.39-3.02 (m,5H), 2.67-2.20 (m, 5H), 2.15-2.00 (m, 2H), 1.90 (h, J=7.0 Hz, 2H),1.50-1.31 (m, 4H), 1.26 (t, J=7.1 Hz, 2H), 1.03 (td, J=7.4, 2.6 Hz, 3H),0.56 (ddd, J=73.4, 8.4, 6.9 Hz, 6H). ppm; ESMS calculated forC₄₉H₅₀N₈O₁₀: 910.4; found: 911.1 (M+H⁺).

SDC-TRAP-0207

(S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl(2-(4-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)piperidin-1-yl)-2-oxoethyl)(methyl)carbamate

¹H NMR (400 MHz, Chloroform-d) δ 8.19 (dd, J=9.2, 2.9 Hz, 1H), 7.95-7.78(m, 1H), 7.71-7.49 (m, 3H), 7.38 (dd, J=28.1, 8.6 Hz, 1H), 7.18-7.05 (m,2H), 6.50 (dd, J=15.3, 3.4 Hz, 1H), 6.37-6.15 (m, 2H), 5.72 (d, J=16.3Hz, 1H), 5.38-5.09 (m, 3H), 4.49-4.02 (m, 5H), 3.78 (dd, J=12.7, 5.5 Hz,1H), 3.27 (s, 2H), 3.23-2.95 (m, 4H), 2.86-2.55 (m, 2H), 2.00-1.68 (m,6H), 1.67-1.48 (m, 2H), 1.47-1.13 (m, 6H), 1.08-0.83 (m, 4H), 0.53-0.19(m, 6H). ppm; ESMS calculated for C₅₂H₅₄N₈O₁₀: 950.4; found: 951.2(M+H⁺).

SDC-TRAP-0206

(S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl(2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenoxy)piperidin-1-yl)-2-oxoethyl)(methyl)carbamate

¹H NMR (400 MHz, Chloroform-d) δ 8.16 (t, J=8.8 Hz, 1H), 7.87 (dd,J=16.2, 2.5 Hz, 1H), 7.69-7.51 (m, 2H), 7.39 (t, J=5.9 Hz, 1H),7.30-7.25 (m, 2H), 7.05 (dd, J=8.6, 5.3 Hz, 2H), 6.59-6.30 (m, 2H), 5.73(dd, J=16.3, 2.6 Hz, 1H), 5.41-5.13 (m, 3H), 4.66 (s, 1H), 4.45-4.16 (m,2H), 4.00-3.77 (m, 1H), 3.71 (d, J=15.5 Hz, 1H), 3.49 (d, J=13.3 Hz,1H), 3.45-3.33 (m, 2H), 3.31 (s, 3H), 3.14 (d, J=9.0 Hz, 3H), 3.01-2.84(m, 1H), 2.03-1.79 (m, 4H), 1.76-1.51 (m, 4H), 1.43-1.32 (m, 3H),1.30-1.14 (m, 3H), 1.02 (td, J=7.4, 3.6 Hz, 3H), 0.98-0.89 (m, 1H), 0.76(dd, J=6.8, 4.1 Hz, 6H). ppm; ESMS calculated for C₅₁H₅₄N₈O₁₁: 954.4;found: 955.2 (M+H⁺).

SDC-TRAP-0205

(S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)piperidine-1-carboxylate

¹H NMR (400 MHz, Chloroform-d) δ 8.20 (d, J=9.2 Hz, 1H), 7.84 (d, J=2.5Hz, 1H), 7.71-7.45 (m, 4H), 7.38 (t, J=5.9 Hz, 1H), 7.26-7.11 (m, 2H),6.61-6.23 (m, 3H), 5.75 (d, J=16.3 Hz, 1H), 5.39-5.17 (m, 3H), 4.55-4.17(m, 4H), 3.49-3.28 (m, 2H), 3.24-2.84 (m, 4H), 2.79 (p, J=6.9 Hz, 1H),2.00-1.77 (m, 6H), 1.65-1.55 (m, 2H), 1.40 (q, J=7.5 Hz, 5H), 1.21 (t,J=7.3 Hz, 3H), 1.03 (t, J=7.3 Hz, 3H), 0.48 (ddd, J=58.3, 7.0, 4.0 Hz,6H). ppm; ESMS calculated for C₅₂H₅₄N₈O₉: 934.4; found: 935.2 (M+H⁺).

SDC-TRAP-0208

(S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)piperidine-1-carboxylate

¹H NMR (400 MHz, DMSO-d₆) δ 10.84 (d, J=12.7 Hz, 1H), 10.08 (d, J=16.6Hz, 1H), 8.75 (s, 1H), 7.75 (dd, J=51.2, 8.9 Hz, 1H), 7.44-7.13 (m, 4H),7.13-6.64 (m, 3H), 6.40-6.02 (m, 3H), 5.35-4.86 (m, 4H), 4.09 (s, 3H),3.56 (s, 1H), 3.05-2.71 (m, 5H), 2.69-2.39 (m, 2H), 2.00-1.85 (m, 2H),1.44 (d, J=84.1 Hz, 5H), 1.14-0.99 (m, 4H), 0.82 (td, J=7.2, 4.4 Hz,3H), 0.71 (q, J=10.2, 8.4 Hz, 4H), 0.32 (dd, J=19.9, 8.4 Hz, 6H). ppm;ESMS calculated for C₅₂H₅₄N₈O₉: 934.4; found: 935.1 (M+H⁺).

¹H NMR (400 MHz, Chloroform-d) δ 11.34 (s, 1H), 8.17-8.05 (m, 1H), 7.85(dt, J=10.0, 2.6 Hz, 1H), 7.78-7.67 (m, 1H), 7.63-7.49 (m, 2H),7.45-7.36 (m, 1H), 7.01 (d, J=8.5 Hz, 2H), 6.43-6.30 (m, 2H), 5.69 (tt,J=14.8, 5.9 Hz, 1H), 5.35-5.14 (m, 3H), 4.90 (d, J=7.9 Hz, 1H), 4.62 (s,1H), 4.14-3.93 (m, 3H), 3.83 (dt, J=9.9, 7.1 Hz, 2H), 3.77-3.65 (m, 2H),3.54 (d, J=12.6 Hz, 1H), 3.43-3.31 (m, 2H), 3.12 (q, J=8.5, 7.0 Hz, 2H),2.99-2.82 (m, 1H), 2.45-2.19 (m, 2H), 2.11 (s, 1H), 2.09-1.99 (m, 2H),1.88 (p, J=6.9 Hz, 2H), 1.75 (s, 2H), 1.44-1.15 (m, 7H), 1.06-0.89 (m,4H), 0.88-0.60 (m, 6H). ESMS calculated for C₅₃H₅₆N₈O₁₁: 980.4; found:980.1 (M+H⁺).

¹H NMR (400 MHz, DMSO-d₆) δ 11.91-11.84 (m, 1H), 9.58-9.46 (m, 2H),8.22-8.13 (m, 1H), 7.97 (d, J=2.6 Hz, 1H), 7.83 (dd, J=4.4, 2.4 Hz, 1H),7.64 (ddd, J=8.2, 5.0, 2.4 Hz, 1H), 7.59-7.30 (m, 6H), 6.99-6.83 (m,2H), 6.68 (d, J=7.8 Hz, 1H), 6.52 (d, J=7.3 Hz, 1H), 6.43 (dt, J=6.4,3.2 Hz, 1H), 6.27-6.19 (m, 1H), 5.44 (s, 2H), 5.31 (d, J=15.6 Hz, 2H),5.02 (q, J=7.9, 6.0 Hz, 1H), 4.83 (d, J=9.7 Hz, 1H), 4.44-4.28 (m, 2H),4.22 (q, J=7.6 Hz, 2H), 4.08-3.91 (m, 4H), 3.73 (q, J=6.7 Hz, 1H), 3.52(dq, J=11.4, 5.5, 4.8 Hz, 1H), 3.10 (ddt, J=49.9, 25.2, 10.0 Hz, 2H),2.84 (ddt, J=32.9, 13.9, 6.6 Hz, 2H), 2.68-2.52 (m, 4H), 2.36 (d, J=8.3Hz, 1H), 1.45 (s, 3H), 1.36-1.06 (m, 3H), 0.93-0.74 (m, 6H). ESMScalculated for C₅₄H₅₆N₈O₁₀: 976.4; found: 977.2 (M+H⁺).

SDC-TRAP-0213

(S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenoxy)piperidine-1-carbonyl)-2-methylpiperidine-1-carboxylate

¹H NMR (400 MHz, Chloroform-d) δ 8.21 (d, J=9.2 Hz, 1H), 7.87 (s, 1H),7.65 (s, 1H), 7.50 (m, 1H), 7.4 (m, 1H), 7.3 (m, 1H), 7.1 (d, J=1.2 Hz,1H), 6.49 (s, 1H), 6.42 (s, 1H), 5.75 (d, J=16.3 Hz, 1H), 5.35-5.24 (m,3H), 4.72 (s, 1H), 4.30 (m, 1H), 4.17-4.02 (m, 2H), 3.60-3.30 (m, 4H),3.16 (q, J=7.8 Hz, 3H), 3.06 (s, 2H), 2.97 (s, 1H), 2.91 (p, J=7.3, 6.9Hz, 1H), 1.90 (m, 5H), 1.72 (d, J=12.6 Hz, 3H), 1.67-1.53 (m, 1H), 1.39(dt, J=13.1, 7.4 Hz, 4H), 1.30-1.16 (m, 6H), 1.03 (t, J=7.4 Hz, 3H),0.99-0.77 (m, 1H), 0.77-0.69 (m, 6H). ppm; ESMS calculated forC₅₅H₆₀N₈O₁₁: 1008.4; found: 1009.4 (M+H+).

SDC-TRAP-0214

(S)—(S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenoxy)piperidine-1-carbonyl)pyrrolidine-1-carboxylate

¹H NMR (400 MHz, DMSO-d₆) δ 10.75 (s, 1H), 10.23 (s, 2H), 9.78 (s, 1H),8.92 (dt, J=11.8, 5.9 Hz, 1H), 7.98-7.90 (m, 1H), 7.41 (tq, J=5.0, 2.6Hz, 2H), 7.36-7.22 (m, 2H), 7.17-6.95 (m, 3H), 6.63-6.50 (m, 1H),6.40-6.30 (m, 1H), 5.48-5.19 (m, 3H), 4.99 (dd, J=8.4, 4.5 Hz, 1H),4.87-4.73 (m, 1H), 4.66-4.57 (m, 1H), 4.02 (tt, J=12.8, 5.5 Hz, 1H),3.50-3.34 (m, 1H), 3.25-3.04 (m, 4H), 2.41-2.32 (m, 1H), 2.16 (d, J=10.8Hz, 2H), 2.13-1.76 (m, 6H), 1.73-1.63 (m, 2H), 1.60-1.46 (m, 1H),1.40-1.14 (m, 3H), 1.10-0.99 (m, 3H), 0.95-0.76 (m, 6H), 0.71 (dd,J=6.8, 2.8 Hz, 3H). ppm; ESMS calculated for C₅₃H₅₆N₈O₁₁: 980.4; found:981.2 (M+H+).

SDC-TRAP-0215

(S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl2-(4-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)piperidine-1-carbonyl)piperidine-1-carboxylate

¹H NMR (400 MHz, Chloroform-d) δ 8.21 (d, J=9.5 Hz, 1H), 7.87 (s, 1H),7.70 (s, 1H), 7.66-7.48 (m, 3H), 7.36 (s, 1H), 7.12 (d, J=31.7 Hz, 2H),6.42 (d, J=60.7 Hz, 2H), 5.71 (d, J=16.5 Hz, 1H), 5.42-5.03 (m, 3H),4.25 (m, 4H), 3.77 (d, J=14.9 Hz, 3H), 3.38 (dt, J=3.3, 1.7 Hz, 3H),3.18 (s, 3H), 2.80-2.50 (m, 2H), 2.28 (t, J=7.7 Hz, 1H), 1.85 (d, J=64.6Hz, 11H), 1.61 (s, 4H), 1.39 (d, J=7.9 Hz, 3H), 1.04 (t, J=7.4 Hz, 3H),0.45 (d, J=21.7 Hz, 6H). ppm; ESMS calculated for C₅₅H₅₈N₈O₁₀: 990.4;found: 991.3 (M+H+).

SDC-TRAP-0216

(S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenoxy)piperidine-1-carbonyl)piperidine-1-carboxylate

¹H NMR (400 MHz, Chloroform-d) δ 8.17 (t, J=9.0 Hz, 1H), 7.84 (d, J=2.6Hz, 1H), 7.73-7.45 (m, 2H), 7.34 (t, J=5.9 Hz, 1H), 7.02 (d, J=8.2 Hz,2H), 6.43 (s, 1H), 6.33 (s, 1H), 5.74 (d, J=16.8 Hz, 1H), 5.44-5.06 (m,5H), 4.62 (s, 1H), 4.29 (d, J=12.8 Hz, 1H), 3.75 (d, J=98.1 Hz, 4H),3.38 (p, J=7.0 Hz, 2H), 3.15 (q, J=7.3 Hz, 2H), 2.90 (s, 1H), 2.03-1.49(m, 11H), 1.46-1.33 (m, 4H), 1.25-1.14 (m, 6H), 1.01 (q, J=7.3 Hz, 3H),0.97-0.80 (m, 1H), 0.74 (d, J=6.5 Hz, 6H). ppm; ESMS calculated forC₅₄H₅₈N₈O₁₁: 994.4; found: 995.4 (M+H+).

SDC-TRAP-0217

(S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-(1-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzyl)piperidine-4-carbonyl)-2-methylpiperazine-1-carboxylate

¹H NMR (400 MHz, Chloroform-d) δ 8.01 (s, 1H), 7.54 (s, 2H), 7.32 (s,3H), 7.19 (s, 3H), 6.45 (dd, J=18.5, 11.0 Hz, 2H), 5.67 (s, 1H), 5.41(s, 1H), 5.14 (s, 1H), 4.07 (tt, J=6.3, 2.8 Hz, 3H), 3.57 (s, 3H), 3.41(d, J=16.0 Hz, 4H), 2.97 (d, J=56.0 Hz, 4H), 2.40-2.19 (m, 2H),1.82-1.50 (m, 5H), 1.50-1.13 (m, 12H), 1.09-0.79 (m, 8H), 0.72 (s, 6H).ppm; ESMS calculated for C₅₅H₆₁N₉O₁₀: 1007.5; found: 1008.5 (M+H⁺).

SDC-TRAP-0218

(S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl(2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenoxy)piperidin-1-yl)-2-oxoethyl)(methyl)carbamate

¹H NMR (400 MHz, Chloroform-d) δ 8.94 (s, 2H), 7.97 (d, J=9.2 Hz, 1H),7.68 (dd, J=22.4, 7.6 Hz, 4H), 7.32 (t, J=2.5 Hz, 1H), 7.18 (s, 1H),7.08 (s, 1H), 6.79-6.68 (m, 1H), 6.47 (d, J=8.8 Hz, 1H), 6.39 (d, J=15.8Hz, 1H), 5.74 (dd, J=16.8, 3.4 Hz, 2H), 5.35 (dd, J=16.7, 2.7 Hz, 2H),5.22 (d, J=3.0 Hz, 2H), 4.93-4.75 (m, 2H), 4.45 (s, 1H), 4.02 (s, 1H),3.64-3.45 (m, 4H), 3.22 (d, J=11.8 Hz, 3H), 3.11-3.02 (m, 3H), 2.95-2.83(m, 2H), 2.24-2.09 (m, 4H), 1.34 (td, J=7.1, 2.3 Hz, 6H), 1.12 (td,J=7.4, 4.3 Hz, 3H), 0.90-0.78 (m, 3H), 0.73 (d, J=6.9 Hz, 6H). ppm; ESMScalculated for C₅₁H₅₄N₈O₁₁: 954.4; found: 955.4 (M+H+).

SDC-TRAP-0027

2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-N-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)-N-methylacetamide

¹H NMR (400 MHz, DMSO-d₆) δ 11.88 (s, 1H), 9.52 (s, 1H), 9.45 (d, J=11.1Hz, 1H), 8.09 (dd, J=13.5, 9.1 Hz, 1H), 7.63-7.41 (m, 5H), 7.33 (dd,J=32.2, 3.0 Hz, 1H), 6.94 (ddd, J=8.7, 3.3, 2.0 Hz, 1H), 6.74 (d, J=13.7Hz, 1H), 6.50 (s, 1H), 6.43 (dd, J=3.1, 0.8 Hz, 1H), 6.23 (d, J=2.1 Hz,1H), 5.44 (s, 2H), 5.33-5.28 (m, 2H), 5.05 (s, 1H), 4.65 (s, 1H), 4.51(d, J=6.3 Hz, 1H), 4.32 (t, J=6.5 Hz, 1H), 3.80 (t, J=6.2 Hz, 1H), 3.65(t, J=6.5 Hz, 1H), 3.15 (dd, J=17.6, 8.3 Hz, 2H), 2.95-2.80 (m, 4H),1.88 (hept, J=7.2 Hz, 2H), 1.28 (q, J=7.5 Hz, 3H), 0.93-0.78 (m, 9H).

ESMS calculated for C₄₆H₄₅N₇O₉: 839.33; Found: 840.1 (M+H)⁺.

SDC-TRAP-0028

2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-N-(2-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethoxy)ethyl)-N-methylacetamide

ESMS calculated for C₄₈H₄₉N₇O₁₀: 883.35; Found: 884.3 (M+H)⁺.

SDC-TRAP-0029

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl(2-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethoxy)ethyl)(methyl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 11.87 (s, 1H), 9.50 (d, J=19.6 Hz, 2H),8.21-8.14 (m, 1H), 7.96 (d, J=9.5 Hz, 1H), 7.64 (d, J=8.3 Hz, 1H), 7.52(s, 1H), 7.43 (d, J=7.0 Hz, 2H), 7.33 (s, 1H), 6.91 (dd, J=15.2, 8.5 Hz,1H), 6.71 (d, J=8.6 Hz, 1H), 6.52 (s, 1H), 6.43 (d, J=13.7 Hz, 1H), 6.23(s, 1H), 5.44 (s, 2H), 5.33 (s, 2H), 4.42-4.36 (m, 2H), 3.77 (d, J=11.5Hz, 2H), 3.69-3.44 (m, 4H), 3.17 (t, J=7.3 Hz, 2H), 3.03 (s, 1H), 2.89(d, J=13.3 Hz, 2H), 1.89 (dq, J=17.0, 9.1, 8.1 Hz, 2H), 1.27 (d, J=10.5Hz, 3H), 0.85-0.74 (m, 9H). ESMS calculated for C₄₇H₄₇N₇O₁₀: 869.34;Found: 870.2 (M+H)⁺.

SDC-TRAP-0037

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl(2-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethoxy)ethyl)(methyl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 11.87 (s, 1H), 10.30 (s, 1H), 9.54 (s, 1H),9.48 (s, 1H), 7.97 (t, J=9.4 Hz, 1H), 7.45-7.25 (m, 4H), 7.00 (d, J=23.6Hz, 1H), 6.92-6.81 (m, 1H), 6.70 (d, J=2.3 Hz, 1H), 6.39 (d, J=3.0 Hz,1H), 6.23 (d, J=3.2 Hz, 1H), 5.45 (s, 2H), 5.28 (s, 1H), 5.21 (d, J=6.9Hz, 1H), 4.53-4.47 (m, 1H), 3.90 (d, J=6.3 Hz, 1H), 3.18-2.97 (m, 6H),2.88 (dt, J=13.9, 7.0 Hz, 2H), 2.70 (s, 3H), 2.18-2.05 (m, 2H), 1.27(dt, J=14.6, 7.3 Hz, 3H), 1.10-0.76 (m, 9H). ESMS calculated forC₄₇H₄₇N₇O₁₀: 869.34; Found: 870.3 (M+H)⁺.

SDC-TRAP-0038

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)(methyl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 11.94 (s, 1H), 10.33 (s, 1H), 9.52 (s, 1H),9.44 (s, 1H), 8.01 (t, J=9.5 Hz, 1H), 7.67 (d, J=8.8 Hz, 1H), 7.55 (d,J=3.0 Hz, 1H), 7.41-7.25 (m, 4H), 7.13-7.08 (m, 1H), 7.04-6.94 (m, 2H),6.73 (dd, J=7.0, 4.4 Hz, 1H), 6.22 (s, 1H), 5.44 (s, 2H), 5.34 (s, 2H),4.56 (s, 1H), 3.91-3.84 (m, 2H), 3.59-3.50 (m, 2H), 2.97-2.83 (m, 2H),2.59 (s, 3H)-2.31 (s, 1H), 2.14 (q, J=7.3 Hz, 2H), 1.30 (t, J=7.5 Hz,3H), 1.01-0.86 (m, 9H). ESMS calculated for C₄₅H₄₃N₇O₉: 825.31; Found:826.4 (M+H)⁺.

SDC-TRAP-0046

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazine-1-carboxylate

¹H NMR (400 MHz, DMSO-d₆) δ 11.95 (s, 1H), 9.62 (s, 1H), 9.43 (s, 1H),8.18 (d, J=9.2 Hz, 1H), 8.00 (d, J=2.4 Hz, 1H), 7.67 (dd, J=9.1, 2.5 Hz,1H), 7.40-7.31 (m, 3H), 7.18 (d, J=7.9 Hz, 2H), 6.80 (s, 1H), 6.53 (s,1H), 6.28 (s, 1H), 5.44 (s, 2H), 5.34 (s, 2H), 3.69-3.46 (m, 4H), 3.19(q, J=7.7 Hz, 2H), 2.99 (p, J=7.0 Hz, 1H), 1.88 (hept, J=7.1 Hz, 2H),1.30 (t, J=7.5 Hz, 3H), 0.97 (d, J=6.9 Hz, 6H), 0.89 (t, J=7.3 Hz, 3H).ESMS calculated for C₄₅H₄₅N₇O₉: 827.33; Found: 828.2 (M+H)⁺.

SDC-TRAP-0047

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazine-1-carboxylate

¹H NMR (400 MHz, DMSO-d₆) δ 11.94 (s, 1H), 10.34 (s, 1H), 9.60 (s, 1H),9.41 (s, 1H), 8.08-8.00 (m, 1H), 7.47-7.39 (m, 2H), 7.32 (d, J=8.0 Hz,3H), 7.15 (d, J=8.1 Hz, 2H), 6.96 (s, 1H), 6.78 (s, 1H), 6.27 (s, 1H),5.44 (d, J=2.6 Hz, 2H), 5.32-5.27 (m, 2H), 3.71 (s, 1H), 3.62 (s, 1H),3.56-3.47 (m, 2H), 3.39 (s, 5H), 3.37-3.23 (m, 6H), 3.09 (q, J=7.5 Hz,2H), 2.97 (p, J=6.9 Hz, 1H), 2.31 (s, 1H), 2.22 (s, 1H), 2.14 (q, J=7.3Hz, 2H), 1.30 (t, J=7.5 Hz, 3H), 1.01-0.86 (m, 9H). ESMS calculated forC₄₅H₄₅N₇O₉: 827.33; Found: 828.3 (M+H)⁺.

SDC-TRAP-0067

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-((4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperidin-1-yl)methyl)piperidine-1-carboxylate

ESMS calculated for C₅₂H₅₇N₇O₉: 923.42; Found: 924.3 (M+H)⁺.

SDC-TRAP-0070

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-(2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenyl)piperazin-1-yl)ethyl)piperidine-1-carboxylate

ESMS calculated for C₅₁H₅₆N₈O₉: 924.42; Found: 925.3 (M+H)⁺.

SDC-TRAP-0077

9-(2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)-2-oxoethoxy)-4,11-diethyl-4-hydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione

¹H NMR (400 MHz, DMSO-d₆) δ 11.93 (s, 1H), 9.61 (s, 1H), 9.41 (s, 1H),8.09 (d, J=9.2 Hz, 1H), 7.53 (dd, J=9.2, 2.7 Hz, 1H), 7.44 (d, J=2.8 Hz,1H), 7.37-7.25 (m, 3H), 7.15 (d, J=8.3 Hz, 2H), 6.78 (s, 1H), 6.51 (s,1H), 6.27 (s, 1H), 5.43 (s, 2H), 5.30 (s, 2H), 5.10 (s, 2H), 3.55 (s,2H), 3.49 (d, J=9.1 Hz, 4H), 3.16 (q, J=7.6 Hz, 2H), 2.97 (p, J=6.9 Hz,1H), 2.46 (d, J=5.8 Hz, 2H), 2.33 (s, 2H), 1.87 (hept, J=7.0 Hz, 2H),1.29 (t, J=7.5 Hz, 3H), 0.98 (d, J=6.9 Hz, 6H), 0.89 (t, J=7.3 Hz, 3H).ESMS calculated for C₄₆H₄₇N₇O₉: 841.34; Found: 842.1 (M+H)⁺.

SDC-TRAP-0079

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-2-fluorobenzyl)piperazine-1-carboxylate

¹H NMR (400 MHz, DMSO-d₆) δ 11.99 (s, 1H), 10.35 (s, 1H), 9.64 (s, 1H),9.40 (s, 1H), 8.03 (d, J=9.1 Hz, 1H), 7.41 (d, J=6.9 Hz, 3H), 7.07 (d,J=10.8 Hz, 1H), 6.97 (d, J=9.8 Hz, 2H), 6.87 (s, 1H), 6.27 (s, 1H), 5.44(s, 2H), 5.29 (s, 2H), 3.73 (d, J=13.4 Hz, 1H), 3.56 (d, J=16.6 Hz, 3H),3.32-3.23 (m, 4H), 3.09 (d, J=8.0 Hz, 2H), 3.05-2.96 (m, 1H), 2.55 (s,1H), 2.39-2.32 (m, 1H), 2.24 (s, 2H), 2.13 (d, J=7.7 Hz, 2H), 1.28 (q,J=13.0, 10.1 Hz, 3H), 0.96 (d, J=6.9 Hz, 6H), 0.89 (t, J=7.3 Hz, 3H).ESMS calculated for C₄₅H₄₄FN₇O₉: 845.32; Found: 846.2 (M+H)⁺.

SDC-TRAP-0081

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl-4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenyl)piperazine-1-carboxylate

¹H NMR (400 MHz, DMSO-d₆) δ 11.94 (s, 1H), 10.38 (s, 1H), 9.66 (s, 1H),9.51 (s, 1H), 7.99 (d, J=9.4 Hz, 1H), 7.46 (d, J=5.6 Hz, 2H), 7.21 (s,1H), 7.12 (d, J=8.5 Hz, 2H), 7.04 (d, J=9.9 Hz, 3H), 6.84 (s, 1H), 6.33(s, 1H), 5.52 (s, 2H), 5.35 (s, 2H), 3.91-3.83 (m, 4H), 3.20-3.09 (m,6H), 3.02 (p, J=7.0 Hz, 1H), 2.23 (q, J=7.3 Hz, 2H), 1.35 (t, J=7.3 Hz,3H), 1.07-0.91 (m, 9H). ESMS calculated for C₄₄H₄₃N₇O₉: 813.31; Found:814.2 (M+H)⁺.

SDC-TRAP-0083

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)isoindoline-2-carboxylate

¹H NMR (400 MHz, DMSO-d₆) δ 12.01 (s, 1H), 9.66 (s, 1H), 9.45 (s, 1H),8.27 (d, J=9.2 Hz, 1H), 8.15 (s, 1H), 7.85-7.77 (m, 1H), 7.48-7.35 (m,3H), 7.15 (d, J=8.0 Hz, 1H), 6.99 (s, 1H), 6.60 (s, 1H), 6.32 (s, 1H),5.50 (s, 2H), 5.41 (s, 2H), 5.03 (d, J=13.8 Hz, 2H), 4.80 (d, J=13.5 Hz,2H), 3.29-3.20 (m, 2H), 3.09 (p, J=7.1 Hz, 1H), 1.94 (hept, J=7.2 Hz,2H), 1.37 (t, J=7.4 Hz, 3H), 1.11 (d, J=6.9 Hz, 6H), 0.95 (t, J=7.3 Hz,3H). ESMS calculated for C₄₂H₃₈N₆O₉: 770.27; Found: 771.2 (M+H)⁺.

SDC-TRAP-0094

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenyl)piperazine-1-carbonyl)piperidine-1-carboxylate

ESMS calculated for C₅₀H₅₂N₈O₁₀: 924.38; Found: 925.1 (M+H)⁺.

SDC-TRAP-0095

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1-methyl-1H-benzo[d]imidazol-2-yl)piperidine-1-carboxylate

¹H NMR (400 MHz, DMSO-d₆) δ 11.87 (s, 1H), 9.53 (s, 1H), 9.34 (s, 1H),8.19 (d, J=9.1 Hz, 1H), 8.04 (d, J=2.6 Hz, 1H), 7.71 (dd, J=9.2, 2.5 Hz,1H), 7.51 (d, J=8.6 Hz, 1H), 7.39 (d, J=1.9 Hz, 1H), 7.34 (s, 1H), 7.05(dd, J=8.6, 2.0 Hz, 1H), 6.87 (s, 1H), 6.54 (s, 1H), 6.21 (s, 1H), 5.45(s, 2H), 5.35 (s, 2H), 4.37 (s, 1H), 4.18 (d, J=12.6 Hz, 1H), 3.83 (s,3H), 3.43-3.28 (m, 4H), 3.27-3.15 (m, 4H), 2.97 (p, J=6.9 Hz, 1H), 1.88(hept, J=7.2 Hz, 2H), 1.31 (t, J=7.6 Hz, 3H), 0.97 (d, J=6.9 Hz, 6H),0.89 (t, J=7.3 Hz, 3H). ESMS calculated for C₄₇H₄₆N₈O₉: 866.34; Found:867.2 (M+H)⁺.

SDC-TRAP-0101

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenyl)piperazin-1-yl)piperidine-1-carboxylate

¹H NMR (400 MHz, DMSO-d₆) δ 11.74 (s, 1H), 9.50 (s, 1H), 9.37 (s, 1H),8.05 (d, J=9.2 Hz, 1H), 7.87 (d, J=2.5 Hz, 1H), 7.55 (dd, J=9.1, 2.5 Hz,1H), 7.20 (s, 1H), 6.90 (d, J=8.8 Hz, 2H), 6.80 (d, J=8.8 Hz, 2H), 6.65(s, 1H), 6.42 (s, 1H), 6.16 (s, 1H), 5.32 (s, 2H), 5.21 (s, 2H), 4.15(s, 1H), 4.00-3.85 (m, 1H), 3.12-3.00 (m, 7H), 2.84 (dq, J=12.6, 6.4,5.9 Hz, 2H), 2.38 (p, J=1.8 Hz, 12H), 1.87 (s, 1H), 1.75 (hept, J=7.0,6.5 Hz, 4H), 1.42 (s, 1H), 1.36 (s, 1H), 1.11 (dt, J=47.7, 7.3 Hz, 3H),0.84 (d, J=6.8 Hz, 6H), 0.76 (t, J=7.2 Hz, 3H). ESMS calculated forC₄₉H₅₂N₈O₉: 896.39; Found: 897.3 (M+H)⁺.

SDC-TRAP-0220

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)-2-methylpiperazine-1-carboxylate

¹H NMR (400 MHz, DMSO-d₆) δ 11.77 (s, 1H), 9.44 (s, 1H), 9.25 (s, 1H),8.01 (d, J=9.1 Hz, 1H), 7.83 (d, J=2.5 Hz, 1H), 7.50 (dd, J=9.1, 2.5 Hz,1H), 7.24-7.14 (m, 3H), 7.01 (d, J=7.9 Hz, 2H), 6.63 (s, 1H), 6.36 (s,1H), 6.11 (s, 1H), 5.27 (s, 2H), 5.17 (s, 2H), 4.18 (s, 1H), 3.41 (d,J=13.7 Hz, 1H), 3.32 (d, J=13.6 Hz, 1H), 3.14 (d, J=11.5 Hz, 3H), 3.03(q, J=7.8 Hz, 2H), 2.82 (p, J=6.9 Hz, 1H), 2.69 (d, J=10.9 Hz, 1H), 2.07(s, 1H), 1.93 (s, 1H), 1.71 (hept, J=7.2 Hz, 2H), 1.24-1.08 (m, 6H),0.80 (d, J=6.9 Hz, 6H), 0.72 (t, J=7.3 Hz, 3H). ESMS calculated forC₄₆H₄₇N₇O₉: 841.34; Found: 842.4 (M+H)⁺.

SDC-TRAP-0010

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-N,1-dimethyl-1H-indole-2-carboxamido)ethyl)(methyl)carbamate

ESMS calculated (C₄₈H₄₈N₈O₁₀): 896.4; found: 897.2 (M+H).

SDC-TRAP-0023

2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-N-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)acetamide

ESMS calculated (C₄₅H₄₃N₇O₉): 825.3; found: 826.2 (M+H).

SDC-TRAP-0024

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl-4-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1-methyl-1H-indole-2-carboxamido)butanoate

¹H NMR (400 MHz, Methanol-d₄) δ 7.88 (d, J=9.1 Hz, 1H), 7.44 (d, J=2.0Hz, 1H), 7.38-7.24 (m, 4H), 7.15 (dd, J=8.8, 2.0 Hz, 1H), 6.74 (s, 1H),6.67 (s, 1H), 6.26 (s, 1H), 5.62 (d, J=16.6 Hz, 1H), 5.44 (d, J=16.7 Hz,1H), 5.05 (d, J=18.7 Hz, 1H), 4.81 (d, J=18.7 Hz, 1H), 3.58 (s, 3H),3.49-3.42 (m, 1H), 3.40-3.32 (m, 1H), 3.10-2.96 (m, 1H), 2.96-2.83 (m,2H), 2.73 (td, J=6.8, 2.5 Hz, 2H), 2.19 (ddt, J=18.2, 14.3, 7.2 Hz, 2H),2.09-1.90 (m, 2H), 1.29 (t, J=7.6 Hz, 3H), 1.01 (t, J=7.4 Hz, 3H), 0.74(dd, J=10.2, 6.8 Hz, 6H); ESMS calculated (C₄₇H₄₅N₇O₁₀): 867.3; found:868.3 (M+H).

SDC-TRAP-0026

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl-4-((2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)amino)-4-oxobutanoate

¹H NMR (400 MHz, Methanol-d₄) δ 8.00-7.88 (m, 2H), 7.42 (d, J=2.0 Hz,1H), 7.37-7.23 (m, 3H), 7.02 (d, J=3.2 Hz, 1H), 6.87 (dd, J=8.7, 2.0 Hz,1H), 6.45 (s, 1H), 6.33 (d, J=3.1 Hz, 1H), 6.23 (s, 1H), 5.61 (d, J=16.7Hz, 1H), 5.44 (d, J=16.6 Hz, 1H), 5.06 (d, J=18.6 Hz, 1H), 4.89 (d,J=18.6 Hz, 1H), 4.58 (s, 1H), 4.08-3.97 (m, 1H), 3.45-3.40 (m, 1H),3.35-3.29 (m, 1H), 2.99-2.74 (m, 5H), 2.51-2.40 (m, 2H), 2.27-2.12 (m,2H), 1.36-1.18 (m, 3H), 1.02 (t, J=7.4 Hz, 3H), 0.58 (dd, J=6.9, 5.1 Hz,6H); ESMS calculated (C₄₇H₄₅N₇O₁₀): 867.3; found: 868.3 (M+H).

SDC-TRAP-0042

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperidin-1-yl)-4-oxobutanoate

¹H NMR (400 MHz, Methanol-d₄) δ 7.99 (d, J=9.5 Hz, 1H), 7.45-7.33 (m,3H), 7.27-7.05 (m, 4H), 6.64 (d, J=8.7 Hz, 1H), 6.26 (s, 1H), 5.60 (dd,J=16.7, 3.0 Hz, 1H), 5.51

-   -   5.40 (m, 1H), 5.24 (d, J=1.5 Hz, 2H), 4.48 (d, J=12.9 Hz, 1H),        3.88 (d, J=13.7 Hz, 1H), 3.34 (s, 2H), 3.13 (q, J=7.4 Hz, 2H),        3.02-2.83 (m, 3H), 2.83-2.63 (m, 3H), 2.55 (d, J=7.0 Hz, 1H),        2.46 (d, J=13.3 Hz, 2H), 2.21 (dp, J=21.6, 7.1 Hz, 2H),        1.70-1.56 (m, 2H), 1.36 (td, J=7.7, 3.6 Hz, 3H), 1.03 (td,        J=7.5, 1.4 Hz, 3H), 0.88-0.79 (m, 6H); ESMS calculated        (C₄₉H₅₀N₆O₁₀): 882.4; found: 883.3 (M+H).

SDC-TRAP-0043

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)-4-oxobutanoate

¹H NMR (400 MHz, Methanol-d₄) δ 7.99 (d, J=8.9 Hz, 1H), 7.43-7.28 (m,5H), 7.26-7.17 (m, 2H), 6.68 (s, 1H), 6.24 (s, 1H), 5.59 (d, J=16.6 Hz,1H), 5.45 (d, J=16.6 Hz, 1H), 5.24 (s, 2H), 3.59 (s, 2H), 3.54-3.31 (m,4H), 3.13 (q, J=7.7 Hz, 2H), 3.02-2.83 (m, 2H), 2.81-2.62 (m, 3H), 2.45(s, 1H), 2.35 (s, 1H), 2.30-2.10 (m, 4H), 1.40 (m, 3H), 1.03 (t, J=7.4Hz, 3H), 0.84 (t, J=6.7 Hz, 6H); ESMS calculated (C₄₈H₄₉N₇O₁₀): 883.3;found: 884.3 (M+H).

SDC-TRAP-0044

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl(4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)butyl)(methyl)carbamate

¹H NMR (400 MHz, Methanol-d₄) δ 8.13 (dd, J=9.9, 7.8 Hz, 1H), 7.93 (d,J=2.7 Hz, 1H), 7.66-7.59 (m, 2H), 7.45-7.40 (m, 2H), 7.26-7.20 (m, 2H),6.66 (d, J=16.5 Hz, 1H), 6.27-6.19 (m, 1H), 5.58 (d, J=16.2 Hz, 1H),5.38 (dd, J=16.2, 1.8 Hz, 1H), 5.27 (s, 2H), 4.85 (s, 1H), 3.64-3.52 (m,3H), 3.48-3.40 (m, 1H), 3.17 (s, 3H), 3.05 (s, 1H), 3.01-2.87 (m, 2H),2.70-2.49 (m, 9H), 1.99-1.91 (m, 2H), 1.80-1.64 (m, 5H), 1.37 (td,J=7.3, 2.1 Hz, 3H), 1.00 (td, J=7.3, 4.3 Hz, 3H), 0.95-0.77 (m, 6H);ESMS calculated (C₅₀H₅₆N₈O₉): 912.4; found: 913.3 (M+H).

SDC-TRAP-0045

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenyl)piperazine-1-carboxylate

¹H NMR (400 MHz, DMSO-d₆) δ 11.88 (s, 1H), 9.62 (s, 1H), 9.46 (s, 1H),8.19 (d, J=9.1 Hz, 1H), 8.04 (d, J=2.6 Hz, 1H), 7.71 (dd, J=9.2, 2.5 Hz,1H), 7.33 (s, 1H), 7.07 (d, J=9.0 Hz, 2H), 7.00 (d, J=9.1 Hz, 2H), 6.82(s, 1H), 6.56 (s, 1H), 6.27 (s, 1H), 5.44 (s, 2H), 5.35 (s, 2H), 3.81(s, 2H), 3.72-3.52 (m, 4H), 3.48-3.19 (m, 4H), 2.99 (p, J=6.8 Hz, 1H),1.87 (dt, J=14.9, 7.0 Hz, 2H), 1.30 (t, J=7.6 Hz, 3H), 0.99 (d, J=6.9Hz, 6H), 0.88 (t, J=7.3 Hz, 3H); ESMS calculated (C₄₄H₄₃N₇O₉): 813.3;found: 814.3 (M+H).

SDC-TRAP-0055

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl(4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)butyl)(methyl)carbamate

To a solution of SN-38 (3 g, 7.65 mmol) in DCM/THF (150 mL/150 mL) wasadded (Boc)₂O (2 g, 9.16 mmol) and pyridine (20 mL). The suspension wasstirred at room temperature until the solution turned clear. Thesolution was diluted with DCM (100 mL) and washed with 2N HCl (100mL×3). The organic phase was collected, dried over Na₂SO₄ andconcentrated. The resulting crude product was used directly for the nextstep without purification.

To the solution of SN-38-¹⁰OBoc (1 g, 2.03 mmol) in DCM (50 mL) wasadded 4-nitrophenyl chloroformate (0.49 g, 2.44 mmol) followed by DMAP(0.74 g, 6.05 mmol). The reaction was stirred at room temperature for 1hr before it was diluted with 100 mL of DCM. The reaction solution waswashed with 0.1 N HCl (50 mL×3), dried over Na₂SO₄ and concentrated. Theresulting solid was washed with Et₂O to remove excess 4-nitrophenylchloroformate. The resulting crude product is used directly for the nextstep without purification.

To the solution of4-(5-hydroxy-4-(4-(piperazin-1-ylmethyl)phenyl)-4H-1,2,4-triazol-3-yl)-6-isopropylbenzene-1,3-diol(0.46 g, 1.12 mmol) in MeOH (10 mL) was added t-butylmethyl(4-oxobutyl)carbamate (0.45 g, 2.23 mmol) and acetic acid (3drops) at room temperature. NaBH₃CN (0.28 g, 4.44 mmol) was added as twoportions in 10 min. The resulting solution was stirred at roomtemperature for 30 min before it was concentrated. Column chromatographygavet-butyl-(4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)butyl)(methyl)carbamate (0.48 g, 72%).

To the solution oft-butyl-(4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)butyl)(methyl)carbamate (0.48 g, 0.81 mmol) in DCM (15mL) was added 4N HCl in dioxane (5 mL). The reaction was stirred at roomtemperature for 3 hr before it was concentrated. The resulting crudeproduct was used directly for the next step without purification.

To the solution of 4-(5-hydroxy-4-(4-((4-(4-(methylamino)butyl)piperazin-1-yl)methyl)phenyl)-4H-1,2,4-triazol-3-yl)-6-isopropylbenzene-1,3-diol(HCl salt, 0.1 g, 0.19 mmol) in DMF (4 mL) was added t-butyl(4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-4,9-diyl)(4-nitrophenyl) dicarbonate (0.16 g, 0.24 mmol) and TEA (0.09 mL, 0.65mmol). The reaction was stirred at room temperature for 2 hr before itwas diluted with H₂O (20 mL) and EtOAc (20 mL). The organic phase wascollected, dried over Na₂SO₄ and concentrated. Column chromatographygave9-((t-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl(4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)butyl)(methyl)carbamate(0.15 g, 75%).

To the solution of9-((t-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl(4-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)butyl)(methyl)carbamate (0.15 g, 0.15 mmol) in DCM(5 mL) was added 4N HCl in dioxane (5 mL). The reaction was stirred atroom temperature for 3 hr before it was concentrated. Columnchromatography gave SDC-TRAP-0055 (0.09 g, 66%) as yellow solid.

¹H NMR (400 MHz, Methanol-d₄) δ 7.93 (dd, J=9.5, 2.8 Hz, 1H), 7.40-7.28(m, 4H), 7.26-7.13 (m, 3H), 6.63 (d, J=6.4 Hz, 1H), 6.17 (s, 1H), 5.48(dd, J=16.7, 11.7 Hz, 1H), 5.41-5.27 (m, 1H), 5.17 (d, J=2.4 Hz, 2H),3.57 (s, 1H), 3.45 (s, 1H), 3.25 (m, 5H), 3.15-3.00 (m, 8H), 2.92 (p,J=6.9 Hz, 3H), 2.75 (s, 1H), 2.10 (dp, J=21.9, 7.3 Hz, 2H), 1.82-1.46(m, 5H), 1.28 (td, J=7.6, 1.9 Hz, 3H), 0.95 (dt, J=13.8, 7.4 Hz, 3H),0.81 (dd, J=7.0, 2.0 Hz, 6H); ESMS calculated (C₅₀H₅₆N₈O₉): 912.4;found: 913.1 (M+H).

SDC-TRAP-0056

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenyl)piperazin-1-yl)acetate

¹H NMR (400 MHz, DMSO-d₆) δ 11.84 (s, 1H), 10.32 (s, 1H), 9.57 (s, 1H),9.44 (s, 1H), 8.00-7.92 (m, 1H), 7.40-7.37 (m, 2H), 6.99-6.97 (m, 3H),6.90-6.83 (m, 2H), 6.76 (s, 1H), 6.25 (s, 1H), 5.50 (s, 2H), 5.30 (d,J=3.5 Hz, 2H), 3.58 (d, J=16.5 Hz, 1H), 3.42 (d, J=16.4 Hz, 1H),3.18-3.07 (m, 6H), 2.95 (p, J=6.8 Hz, 1H), 2.65 (t, J=5.2 Hz, 4H), 2.15(dt, J=9.4, 6.5 Hz, 2H), 1.29 (t, J=7.5 Hz, 3H), 0.93 (dd, J=6.8, 1.8Hz, 9H); ESMS calculated (C₄₅H₄₅N₇O₉): 827.3; found: 828.0 (M+H).

SDC-TRAP-0057

9-(2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenyl)piperazin-1-yl)ethoxy)-4,11-diethyl-4-hydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione

ESMS calculated (C₄₅H₄₇N₇O₈): 813.3; found: 814.1 (M+H).

SDC-TRAP-0058

9-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethoxy)-4,11-diethyl-4-hydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione

ESMS calculated (C₄₃H₄₀N₆O₈): 768.3; found: 769.1 (M+H).

SDC-TRAP-0060

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-(3-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenyl)propanoyl)piperazine-1-carboxylate

ESMS calculated (C₄₇H₄₇N₇O₁₀): 869.3; found: 870.0 (M+H).

SDC-TRAP-0061

9-(3-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenyl)piperazin-1-yl)propoxy)-4,11-diethyl-4-hydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione

ESMS calculated (C₄₆H₄₉N₇O₈): 827.3; found: 828.1 (M+H).

SDC-TRAP-0071

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperidin-1-yl)acetate

¹H NMR (400 MHz, Methanol-d₄) δ 7.86 (d, J=9.1 Hz, 1H), 7.32-7.21 (m,2H), 7.18 (s, 1H), 7.15-7.06 (m, 2H), 7.06-6.98 (m, 2H), 6.49 (s, 1H),6.16 (s, 1H), 5.52 (d, J=16.7 Hz, 1H), 5.35 (d, J=16.7 Hz, 1H), 5.08 (s,2H), 3.49-3.31 (m, 2H), 2.99 (q, J=7.6 Hz, 2H), 2.87-2.66 (m, 3H), 2.42(d, J=6.9 Hz, 2H), 2.21-2.00 (m, 4H), 1.54-1.33 (m, 3H), 1.28-1.15 (m,5H), 0.93 (t, J=7.4 Hz, 3H), 0.66 (t, J=7.1 Hz, 6H); ESMS calculated(C₄₇H₄₈N₆O₉): 840.3; found: 841.2 (M+H).

SDC-TRAP-0072

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1-methyl-1H-indole-2-carbonyl)piperazine-1-carboxylate

¹H NMR (400 MHz, DMSO-d₆) δ 11.88 (s, 1H), 9.55 (s, 1H), 9.38 (s, 1H),8.20 (d, J=9.1 Hz, 1H), 8.03 (d, J=2.6 Hz, 1H), 7.70 (dd, J=9.2, 2.5 Hz,1H), 7.56-7.49 (m, 2H), 7.33 (s, 1H), 7.03 (dd, J=8.7, 2.1 Hz, 1H), 6.84(s, 1H), 6.76 (s, 1H), 6.54 (s, 1H), 6.21 (s, 1H), 5.44 (s, 2H), 5.35(s, 2H), 3.79 (brs, 7H), 3.60 (s, 2H), 3.25-3.14 (m, 3H), 2.95 (p, J=7.0Hz, 1H), 1.95-1.79 (m, 3H), 1.30 (t, J=8.0 Hz, 3H), 0.94-0.85 (m, 9H);ESMS calculated (C₄₈H₄₆N₈O₁₀): 894.3; found: 895.0 (M+H).

SDC-TRAP-0073

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-((5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1-methyl-1H-indol-2-yl)methyl)piperazine-1-carboxylate

ESMS calculated (C₄₈H₄₈N₈O₉): 880.4; found: 881.1 (M+H).

SDC-TRAP-0074

9-acetoxy-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazine-1-carboxylate

¹H NMR (400 MHz, DMSO-d₆) δ 11.94 (s, 1H), 9.61 (s, 1H), 9.42 (s, 1H),8.21 (d, J=9.2 Hz, 1H), 8.03 (s, 1H), 7.68 (d, J=9.1 Hz, 1H), 7.32 (d,J=7.9 Hz, 2H), 7.14 (d, J=8.0 Hz, 2H), 7.05 (s, 1H), 6.78 (s, 1H), 6.26(s, 1H), 5.46 (d, J=4.8 Hz, 2H), 5.35 (s, 2H), 3.73 (s, 1H), 3.62 (s,1H), 3.52-3.44 (m, 3H), 3.28-3.13 (m, 4H), 2.97 (p, J=7.1 Hz, 1H), 2.38(s, 3H), 2.30 (s, 1H), 2.24-2.10 (m, 4H), 1.28 (t, J=7.3 Hz, 3H), 0.92(dd, J=19.9, 7.5 Hz, 9H); ESMS calculated (C₄₇H₄₇N₇O₁₀): 880.4; found:881.1 (M+H).

SDC-TRAP-0075

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-((5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1-methyl-1H-indol-2-yl)methyl)piperazine-1-carboxylate

ESMS calculated (C₄₈H₄₈N₈O₉): 880.4; found: 881.2 (M+H).

SDC-TRAP-0076

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl1-(1-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-0)benzyl)piperidine-4-carbonyl)piperidine-4-carboxylate

To the solution of SN-38-¹⁰OBoc (0.85 g, 1.73 mmol) in DCM (50 mL) wasadded 1-(t-butoxycarbonyl)piperidine-4-carboxylic acid (0.48 g, 2.09mmol) followed by DMAP (0.42 g, 3.44 mmol) and EDC (1 g, 5.2 mmol). Thereaction was stirred at room temperature for 1 hr before it was dilutedwith DCM (100 mL). The organic phase was washed with 2N HCl (50 mL×2),dried over Na₂SO₄ and concentrated. Column chromatography gave4-(9-((t-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)1-t-butyl piperidine-1,4-dicarboxylate (1.03 g, 85%).

To the solution of4-(9-((t-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)1-t-butyl piperidine-1,4-dicarboxylate (1.03 g, 1.46 mmol) in DCM (15mL) was added 4N HCl in dioxane (10 mL). The reaction was heated at 45°C. for 30 min before it was concentrated. The resulting crude product isused directly for the next step without purification.

The suspension of4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-ylpiperidine-4-carboxylate (HCl salt, 0.35 g, 0.65 mmol) in DMF and TEA(20 mL/3 mL) was heated until it turned clear. To the resulting solutionwas added1-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzyl)piperidine-4-carboxylicacid (0.3 g, 0.6 mmol), EDC (0.35 g, 1.82 mmol), and HOBt (Cat.). Thereaction was stirred at room temperature overnight before it was dilutedwith EtOAc (30 mL) and NH₄Cl (20 mL). The organic phase was collected,dried over Na₂SO₄ and concentrated. Column chromatography gaveSDC-TRAP-0076 as a light yellow solid (0.28 g, 47%).

¹HNMR (400 MHz, DMSO-d₆) δ 10.63 (s, 1H), 10.32 (s, 1H), 9.75 (s, 1H),8.94 (t, J=5.9 Hz, 1H), 8.01 (d, J=9.0 Hz, 1H), 7.45-7.34 (m, 4H),7.33-7.26 (m, 2H), 6.93 (s, 1H), 6.56 (s, 1H), 6.34 (s, 1H), 5.49 (s,2H), 5.29 (d, J=2.2 Hz, 2H), 4.14 (s, 1H), 3.87 (s, 1H), 3.47 (s, 2H),3.25-3.05 (m, 4H), 2.92-2.82 (m, 5H), 2.59 (s, 1H), 2.22-2.11 (m, 2H),2.04-1.88 (m, 4H), 1.56 (s, 5H), 1.27 (dd, J=16.8, 9.1 Hz, 5H), 1.03 (t,J=7.2 Hz, 3H), 0.97-0.83 (m, 3H), 0.79 (d, J=6.6 Hz, 6H); ESMScalculated (C₅₅H₆₀N₈O₁₀): 992.4; found: 993.5 (M+H).

SDC-TRAP-0097

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3 ‘,4’:6,7]indolizino[1,2-b]quinolin-4-yl1-(2-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenyl)acetyl)piperidine-4-carboxylate

¹H NMR (400 MHz, Methanol-d₄) δ 7.91 (d, J=9.5 Hz, 1H), 7.31 (d, J=7.7Hz, 2H), 7.23 (t, J=5.6 Hz, 2H), 7.15 (d, J=4.2 Hz, 1H), 7.04 (dd,J=27.7, 8.1 Hz, 2H), 6.12 (d, J=6.1 Hz, 1H), 5.51 (d, J=16.4 Hz, 1H),5.42-5.31 (m, 1H), 5.15 (d, J=15.5 Hz, 2H), 4.50 (s, 3H), 4.04 (s, 1H),3.76 (s, 2H), 3.69 (d, J=16.0 Hz, 2H), 3.25 (s, 6H), 3.06 (d, J=13.2 Hz,5H), 2.81 (d, J=13.5 Hz, 2H), 2.17-2.07 (m, 2H), 1.80 (s, 1H), 1.60 (s,2H), 1.27 (q, J=7.8 Hz, 3H), 1.19 (s, 2H), 0.92 (q, J=6.8 Hz, 3H),0.85-0.68 (m, 7H); ESMS calculated (C₄₇H₄₆N₆O₁₀): 854.3; found: 855.2(M+H).

SDC-TRAP-0100

4,11-Diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl3-(1-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzyl)-N-methylpiperidine-4-carboxamido)propanoate

ESMS calculated (C₅₃H₅₈N₈O₁₀): 966.4; found: 967.4 (M+H).

SDC-TRAP-0111

4,11-Diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl1-(2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenyl)piperazin-1-yl)acetyl)piperidine-4-carboxylate

ESMS calculated (C₅₁H₅₄N₈O₁₀): 938.4; found: 939.4 (M+H).

SDC-TRAP-0112

4,11-Diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl(2-(5-(2,4-dihydroxy-5-isopropylphenyl)-4-(pyridin-3-yl)-4H-1,2,4-triazole-3-carboxamido)ethyl)(methyl)carbamate

ESMS calculated (C₄₃H₄₂N₈O₉): 814.3; found: 815.2 (M+H).

SDC-TRAP-0113

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl1-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzoyl)piperidine-4-carboxylate

¹H NMR (400 MHz, DMSO-d₆) δ 10.33 (s, 2H), 9.73 (s, 1H), 8.98 (t, J=6.0Hz, 1H), 7.99 (s, 1H), 7.48-7.35 (m, 6H), 6.95 (s, 1H), 6.66 (s, 1H),6.32 (s, 1H), 5.49 (s, 2H), 5.29 (d, J=2.6 Hz, 2H), 4.25 (s, 1H), 3.54(s, 1H), 3.42-2.90 (m, 10H), 2.15 (t, J=7.7 Hz, 2H), 1.61 (s, 2H), 1.29(t, J=7.6 Hz, 3H), 1.04 (t, J=7.2 Hz, 3H), 0.93 (t, J=7.4 Hz, 3H), 0.85(d, J=6.8 Hz, 6H); ESMS calculated (C₄₉H₄₉N₇O₁₀): 895.4; found: 896.3(M+H).

SDC-TRAP-0154

4,11-Diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl1-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenoxy)benzoyl)piperidine-4-carboxylate

¹H NMR (400 MHz, DMSO-d₆) δ 10.41 (s, 1H), 10.34 (s, 1H), 9.76 (s, 1H),8.98 (t, J=6.0 Hz, 1H), 8.00 (d, J=9.0 Hz, 1H), 7.49-7.33 (m, 6H),7.14-7.01 (m, 4H), 6.95 (s, 1H), 6.68 (s, 1H), 6.34 (s, 1H), 5.49 (s,2H), 5.30 (s, 2H), 3.18 (p, J=6.9 Hz, 4H), 3.08 (d, J=7.3 Hz, 3H), 2.95(dd, J=15.7, 8.7 Hz, 3H), 2.16 (q, J=7.4 Hz, 2H), 1.96 (s, 2H), 1.60 (s,2H), 1.28 (t, J=7.5 Hz, 3H), 1.05 (t, J=7.1 Hz, 3H), 0.92 (dd, J=11.6,7.0 Hz, 9H); ESMS calculated (C₅₅H₅₃N₇O₁₁): 987.4; found: 988.4 (M+H).

SDC-TRAP-0169

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl3-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenoxy)-N-methylbenzamido)propanoate

ESMS calculated (C₅₃H₅₁N₇O₁₁): 961.4; found: 962.3 (M+H).

SDC-TRAP-0172

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl(1-(1-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzyl)piperidine-4-carbonyl)piperidin-4-yl)(methyl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 10.62 (s, 1H), 9.77 (s, 1H), 8.97 (t, J=5.9Hz, 1H), 8.18 (d, J=9.2 Hz, 1H), 8.01 (s, 1H), 7.68 (dd, J=9.2, 2.4 Hz,1H), 7.39 (d, J=8.2 Hz, 2H), 7.35-7.27 (m, 3H), 6.56 (d, J=17.5 Hz, 2H),6.35 (s, 1H), 5.44 (s, 2H), 5.35 (s, 2H), 4.56 (s, 1H), 4.07 (s, 1H),3.50 (s, 2H), 3.31 (s, 4H), 3.20-3.13 (m, 4H), 3.00 (s, 2H), 2.95-2.83(m, 4H), 2.68-2.60 (m, 2H), 2.04 (s, 2H), 1.87 (dt, J=14.8, 7.1 Hz, 3H),1.61 (s, 5H), 1.30 (t, J=8.0 Hz, 3H), 1.04 (t, J=7.2 Hz, 3H), 0.88 (t,J=8.0 Hz, 3H), 0.81 (d, J=8.0 Hz, 6H); ESMS calculated (C₅₆H₆₃N₉O₁₀):1021.5; found: 1022.5 (M+H).

SDC-TRAP-0180

4,11-Diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl(1-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenoxy)benzoyl)piperidin-4-yl)(methyl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 10.42 (s, 1H), 9.77 (s, 1H), 8.98 (t, J=5.9Hz, 1H), 8.18 (d, J=9.1 Hz, 1H), 8.01 (d, J=2.5 Hz, 1H), 7.68 (dd,J=9.1, 2.4 Hz, 1H), 7.53 (d, J=8.1 Hz, 2H), 7.44-7.35 (m, 2H), 7.33 (s,1H), 7.16-7.06 (m, 4H), 6.69 (s, 1H), 6.53 (s, 1H), 6.35 (s, 1H), 5.44(s, 2H), 5.34 (s, 2H), 4.62-4.22 (m, 2H), 3.77 (s, 1H), 3.26-3.14 (m,5H), 3.05 (s, 2H), 2.98 (p, J=6.9 Hz, 1H), 2.90 (s, 2H), 1.91-1.80 (m,6H), 1.34-1.21 (m, 3H), 1.07 (t, J=7.2 Hz, 3H), 0.93 (d, J=15.2, 8.0 Hz,6H), 0.88 (t, J=8.0 Hz, 3H); ESMS calculated (C₅₆H₅₆N₈O₁₁): 1016.4;found: 1017.5 (M+H).

SDC-TRAP-0181

4-(((4-(4-(4-(3-(2,4-Dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazin-1-yl)butyl)(methyl)carbamoyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-ylacetate

ESMS calculated (C₅₂H₅₈N₈O₁₀): 954.4; found: 955.3 (M+H).

SDC-TRAP-0184

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl(1-(3-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)propanoyl)piperidin-4-yl)(methyl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 11.83 (s, 1H), 9.51 (s, 1H), 9.45 (s, 1H),8.17 (d, J=9.1 Hz, 1H), 7.99 (s, 1H), 7.70-7.62 (m, 1H), 7.54-7.38 (m,3H), 7.32 (s, 1H), 6.95 (dd, J=8.7, 2.0 Hz, 1H), 6.74 (s, 1H), 6.50 (s,1H), 6.42 (d, J=3.1 Hz, 1H), 6.23 (s, 1H), 5.44 (s, 2H), 5.34 (s, 2H),4.53 (s, 1H), 4.43 (t, J=6.8 Hz, 2H), 3.83 (s, 1H), 3.29 (s, 3H),3.22-3.14 (m, 3H), 2.93-2.66 (s, 7H), 1.87 (p, J=7.1 Hz, 2H), 1.49 (s,2H), 1.29 (t, J=8.0 Hz, 3H), 0.92-0.82 (m, 9H); ESMS calculated(C₅₁H₅₂N₈O₁₀): 936.4; found: 937.0 (M+H).

SDC-TRAP-0185

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-(1-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzyl)piperidine-4-carbonyl)-2-methylpiperazine-1-carboxylate

¹H NMR (400 MHz, DMSO-d₆) δ 10.64 (d, J=1.8 Hz, 1H), 9.77 (s, 1H), 8.96(t, J=5.9 Hz, 1H), 8.20 (d, J=9.2 Hz, 1H), 8.03 (d, J=2.5 Hz, 1H), 7.70(dd, J=9.2, 2.5 Hz, 1H), 7.40 (d, J=8.2 Hz, 2H), 7.37-7.24 (m, 3H), 6.59(s, 1H), 6.52 (s, 1H), 6.36 (s, 1H), 5.45 (s, 2H), 5.35 (s, 2H), 4.29(d, J=17.9 Hz, 2H), 4.15-3.81 (m, 2H), 3.51 (s, 2H), 3.27-3.12 (m, 5H),2.95-2.88 (m, 5H), 2.07 (s, 2H), 1.96-1.79 (m, 2H), 1.71-1.63 (m, 5H),1.37-1.13 (m, 6H), 1.05 (t, J=7.2 Hz, 3H), 0.89 (t, J=7.3 Hz, 3H), 0.82(d, J=6.9 Hz, 6H). ESMS calculated (C₅₅H₆₁N₉O₁₀): 1007.5; found: 1008.3(M+H).

SDC-TRAP-0186

4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)methyl)piperidine-1-carboxylate

¹H NMR (400 MHz, DMSO-d₆) δ 11.91 (s, 1H), 9.57 (d, J=4.4 Hz, 2H), 8.17(d, J=9.1 Hz, 1H), 7.97 (d, J=2.5 Hz, 1H), 7.69-7.56 (m, 2H), 7.46 (dd,J=4.9, 2.6 Hz, 2H), 7.32 (s, 1H), 6.98 (dd, J=8.7, 2.0 Hz, 1H), 6.67 (s,1H), 6.53 (s, 1H), 6.47 (d, J=3.1 Hz, 1H), 6.25 (s, 1H), 5.44 (s, 2H),5.34 (s, 2H), 4.25-4.07 (m, 4H), 3.22-3.14 (m, 2H), 3.01 (s, 1H),2.88-2.85 (m, 2H), 2.09 (s, 1H), 1.87 (dt, J=14.7, 7.0 Hz, 2H), 1.58 (d,J=12.2 Hz, 2H), 1.33-1.21 (m, 5H), 0.88 (t, J=7.3 Hz, 3H), 0.77 (d,J=6.9 Hz, 6H); ESMS calculated (C₄₈H₄₇N₇O₉): 865.3; found: 866.0 (M+H).

SDC-TRAP-0201

4,11-Diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl4-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)acetyl)-2-methylpiperazine-1-carboxylate

ESMS calculated (C₄₉H₄₈N₈O₁₀): 908.3; found: 909.0 (M+H).

SDC-TRAP-0202

4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl(2-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)benzoyl)piperazin-1-yl)-2-oxoethyl)carbonate

ESMS calculated (C₅₀H₅₀N₈O₁₂): 954.4; found: 955.1 (M+H).

SDC-TRAP-0203

4,11-Diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl(1-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenoxy)benzoyl)piperidin-4-yl)carbonate

To a solution of tert-butyl 4-hydroxypiperidine-1-carboxylate (0.2 g,1.0 mmol) in THF (4 mL) was added phosgene (15% wt in toluene, 0.66 mL).The reaction was stirred at room temperature for 1 hr. SN-38-¹⁰OBoc (0.2g, 0.4 mmol) was added to the reaction solution, followed by DMAP (0.15g, 1.2 mmol). The reaction was stirred at room temperature for 5 hr. Thereaction was quenched with saturated NH₄Cl (10 mL) and extracted withEtOAc (15 mL×3). The organic phases were combined, dried over Na₂SO₄ andconcentrated. Column chromatography gave tert-butyl4-((((9-((tert-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)oxy)piperidine-1-carboxylate(0.21 g, 73%).

To the solution of4-((((9-((tert-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)oxy)piperidine-1-carboxylate(0.2 g, 0.28 mmol) in DCM/MeOH (5 mL/4 mL) was added 4N HCl in dioxane(5 mL). The reaction was stirred at room temperature for 2 hr before itwas concentrated. The resulting solid was dissolved in DMF (4 mL), and4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethoxycarbonyl)-4H-1,2,4-triazol-4-yl)phenoxy)benzoicacid (0.14 g, 0.28 mmol), EDC (0.16 g, 0.83 mmol), TEA (1 mL), and HOBt(Cat.) were added. The reaction was stirred at room temperatureovernight. The reaction was quenched with saturated NH₄Cl (10 mL) andextracted with EtOAc (15 mL×3). The combined organic phase was driedover Na₂SO₄ and concentrated. Column chromatography gave SDC-TRAP-0203(0.15 g, 54%). ESMS calculated (C₅₅H₅₃N₇O₁₂): 1003.4; found: 1004.5(M+H).

SDC-TRAP-0221

4,11-Diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl(1-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenoxy)benzoyl)piperidin-4-yl)(ethyl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 10.43 (s, 1H), 9.80 (s, 1H), 8.97 (t, J=5.8Hz, 1H), 8.19 (d, J=9.2 Hz, 1H), 8.00 (d, J=2.5 Hz, 1H), 7.67 (dd,J=9.2, 2.4 Hz, 1H), 7.52 (d, J=8.1 Hz, 2H), 7.43-7.31 (m, 3H), 7.16-7.05(m, 4H), 6.68 (s, 1H), 6.54 (s, 1H), 6.35 (s, 1H), 5.44 (s, 2H), 5.34(s, 2H), 4.59 (s, 1H), 4.13 (s, 1H), 3.52-3.35 (m, 4H), 3.20 (dt,J=13.1, 6.8 Hz, 4H), 2.98 (p, J=6.9 Hz, 1H), 1.93-1.80 (m, 6H), 1.30 (t,J=7.5 Hz, 6H), 1.22-1.13 (m, 1H), 1.07 (t, J=7.2 Hz, 3H), 0.96-0.84 (m,9H); ESMS calculated (C₅₇H₅₈N₈O₁₁): 1030.4; found: 1031.5 (M+H).

SDC-TRAP-0222

4,11-Diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl(1-(1-((4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenyl)sulfonyl)piperidine-4-carbonyl)piperidin-4-yl)(methyl)carbamate

¹H NMR (400 MHz, DMSO-d₆) δ 9.91 (s, 1H), 9.69 (s, 1H), 9.05 (t, J=6.0Hz, 1H), 8.18 (d, J=9.2 Hz, 1H), 8.00 (d, J=2.7 Hz, 1H), 7.81-7.73 (m,2H), 7.67 (dd, J=9.2, 2.4 Hz, 1H), 7.59-7.52 (m, 2H), 7.32 (s, 1H), 6.74(s, 1H), 6.51 (s, 1H), 6.28 (s, 1H), 5.75 (s, 1H), 5.44 (s, 2H), 5.34(s, 2H), 4.53 (s, 1H), 4.06 (s, 2H), 3.70 (s, 2H), 3.25-3.14 (m, 6H),3.02-2.93 (m, 3H), 2.84 (s, 1H), 2.67-2.32 (m, 3H), 1.87 (p, J=7.0 Hz,2H), 1.74-1.55 (m, 7H), 1.29 (t, J=8.0 Hz, 3H), 1.08 (t, J=7.2 Hz, 3H),0.95 (d, J=8.0 Hz, 6H), 0.88 (t, J=8.0 Hz, 3H); ESMS calculated(C₅₅H₆₁N₉O₁₂S): 1071.4; found: 1072.6 (M+H).

in vitro activity was determined for these compounds using the HER2degradation assay set forth herein:

HER2 degradation SDC-TRAP-# IC₅₀ (nM) SDC-TRAP-0016 >5000 SDC-TRAP-0027 >5000  SDC-TRAP-0028 >5000  SDC-TRAP-0030 >5000 SDC-TRAP-0031 1270  SDC-TRAP-0022 >5000  SDC-TRAP-0023 4300 SDC-TRAP-0010 >5000  SDC-TRAP-0038 >5000  SDC-TRAP-0037 2112 SDC-TRAP-0026 1780  SDC-TRAP-0029 1373  SDC-TRAP-0046 246 SDC-TRAP-00421057  SDC-TRAP-0043 2135  SDC-TRAP-0047 875 SDC-TRAP-0044 602SDC-TRAP-0045 464 SDC-TRAP-0054 1469  SDC-TRAP-0059 184SDC-TRAP-0014 >5000  SDC-TRAP-0012 >5000  SDC-TRAP-0011 >5000 SDC-TRAP-0055 402 SDC-TRAP-0056 1271  SDC-TRAP-0057 449 SDC-TRAP-00582929  SDC-TRAP-0060 >5000  SDC-TRAP-0063 793 SDC-TRAP-0067 196SDC-TRAP-0070 263 SDC-TRAP-0064 1129  SDC-TRAP-0065 661 SDC-TRAP-0071307 SDC-TRAP-0072 >5000  SDC-TRAP-0073 478 SDC-TRAP-0077 2791 SDC-TRAP-0079 1430  SDC-TRAP-0081 622 SDC-TRAP-0083 1438  SDC-TRAP-0094<78 953    SDC-TRAP-0086 >5,000   SDC-TRAP-0084 1132 SDC-TRAP-0095 >5000  SDC-TRAP-0101 280 SDC-TRAP-0087 535 SDC-TRAP-00904599  SDC-TRAP-0089 1466  SDC-TRAP-0088 221 SDC-TRAP-0074 4120 SDC-TRAP-0075 953 SDC-TRAP-0076 <78 227    SDC-TRAP-0097 >5,000  SDC-TRAP-0091 >5000  SDC-TRAP-0104 350 SDC-TRAP-0092 4706  SDC-TRAP-0100 80 SDC-TRAP-0111 >5000  SDC-TRAP-0112 >5000  SDC-TRAP-0154 191SDC-TRAP-0145 183 SDC-TRAP-0146 1295  SDC-TRAP-0169 611 SDC-TRAP-01613694  SDC-TRAP-0172 <78 56   SDC-TRAP-0180 325 SDC-TRAP-0181 164SDC-TRAP-0185  38 SDC-TRAP-0186 1,619   SDC-TRAP-0184 4,002  SDC-TRAP-0205 564 SDC-TRAP-0206 321 SDC-TRAP-0207 >5,000  SDC-TRAP-0204 >10,000    SDC-TRAP-0208 480 SDC-TRAP-0209 1,130  SDC-TRAP-0210 >10,000    SDC-TRAP-0213 248 SDC-TRAP-0212 2,294  SDC-TRAP-0201 4,670   SDC-TRAP-0202 >5,000   SDC-TRAP-0214 >5,000  SDC-TRAP-0215 2,746   SDC-TRAP-0220 474 445    SDC-TRAP-0203 446

Hsp90^(α) Binding Assay

Binding No SDC-TRAP-# EC₅₀ (nM) 1 SDC-TRAP-0045 96.6 2 SDC-TRAP-0046101.8 3 SDC-TRAP-0063 157.5 4 SDC-TRAP-0064 122.2 5 SDC-TRAP-0184 86.626 SDC-TRAP-0204 82.59 7 SDC-TRAP-0209 54.59 8 SDC-TRAP-0210 91.03

Mouse Plasma Stability Data

% Remaining SDC-TRAP-# (1 h, 37° C.) SDC-TRAP-0022  21% SDC-TRAP-0028 41% SDC-TRAP-0029  47% SDC-TRAP-0037  95% SDC-TRAP-0044  61%SDC-TRAP-0045  45% SDC-TRAP-0046  52% SDC-TRAP-0054 41.0% SDC-TRAP-0071 102% SDC-TRAP-0076  96% SDC-TRAP-0104 95.5% SDC-TRAP-0063 11.1%SDC-TRAP-0064 91.5% SDC-TRAP-0172 74.7% SDC-TRAP-0180 72.4%SDC-TRAP-0184 18.0% SDC-TRAP-0185 68.1% SDC-TRAP-0186 57.9%SDC-TRAP-0042  74% SDC-TRAP-0047  89% SDC-TRAP-0055  103% SDC-TRAP-0056 78% SDC-TRAP-0059  51% SDC-TRAP-0145 14.1% SDC-TRAP-0203 71.2%SDC-TRAP-0215 77.2% SDC-TRAP-0216 67.7% SDC-TRAP-0220 78.3%SDC-TRAP-0202 21.2% SDC-TRAP-0205 58.4% SDC-TRAP-0206 68.6%SDC-TRAP-0208 86.1% SDC-TRAP-0209 67.1% SDC-TRAP-0213 74.7%

Tissue Distribution Data for SDC-TRAP-0045

Tumor Conc. Plasma Conc. (μM) (nmol/g of tissue) Tumor/Plasma Ratio SDC-SDC- SDC- SDC- SDC- SDC- Analyte TRAP- TRAP- TRAP- TRAP- TRAP- TRAP-Target 0045 0053 SN-38 0045 0053 SN-38 0045 0053 SN-38 Time (h) 0.083689 2.70 0.0716 4.30 0.0461 0.344 0.00624 0.0171 4.80 6 1.88 0.2890.00471 2.55 0.590 0.473 1.35 2.04 101 12 0.141 0.0953 BQL 1.13 0.7800.229 8.02 8.18 — 24 0.0113 0.0464 BQL BQL 0.0622 0.0596 — 1.34 — 48 BQL0.00618 BQL BQL 0.764 BQL — 124 —

Tissue Distribution Data for SDC-TRAP-0046

Tumor Conc. Plasma Conc. (μM) (nmol/g of tissue) Tumor/Plasma Ratio SDC-SDC- SDC- SDC- SDC- SDC- Analyte TRAP- TRAP- TRAP- TRAP- SN- TRAP- TRAP-Target 0046 0052 SN-38 0046 0052 38 0046 0052 SN-38 Time (h) 0.083 3600.0782 2.29 6.94 BQL 0.298 0.0193 — 0.130 6 5.88 0.0917 0.0773 4.970.241 0.448 0.844 2.63 5.80 12 2.37 0.0612 0.0389 5.21 0.407 0.344 2.206.65 8.83 24 0.0542 0.0364 0.00955 2.19 1.71 1.01 40.3 46.9 105 48 BQL0.0107 BQL 0.188 1.01 BQL — 94.4 —

Tissue Distribution Data for SDC-TRAP-0056

Tumor Conc. Plasma Conc. (μM) (nmol/g of tissue) Tumor/Plasma Ratio SDC-SDC- SDC- SDC- SDC- SDC- Analyte TRAP- TRAP- TRAP- TRAP- TRAP- TRAP-Target 0056 0096 SN-38 0056 0096 SN-38 0056 0096 SN-38 Time (h) 0.0831220 274 134 6.40 1.654 1.18 0.00525 0.00604 0.00881 6 2.06 0.510 0.4832.65 0.726 0.490 1.28 1.42 1.02 12 0.382 0.151 0.176 0.746 0.252 0.1521.95 1.67 0.86 24 0.0343 0.0130 0.0235 BQL BQL 0.105 — — 4.48 48 BQL BQLBQL BQL 0.0581 0.0259 — — —

Tissue Distribution Data for SDC-TRAP-0063

Tumor Conc. Plasma Conc. (μM) (nmol/g of tissue) Tumor/Plasma Ratio SDC-SDC- SDC- Analyte TRAP- TRAP- TRAP- Target 0063 DP-1 SN-38 0063 DP-1SN-38 0063 DP-1 SN-38 Time (h) 0.083 526 0.0662 20.4 6.43 0.00758 1.470.0122 0.114 0.0721 6 1.69 0.0397 0.0509 1.61 0.111 0.730 0.958 2.7914.3 24 0.00675 0.0175 0.0240 0.203 0.404 0.618 30.1 23.1 25.8 48 BQL0.00793 0.00524 0.0188 1.06 0.296 — 134 56.4

Tissue Distribution Data for SDC-TRAP-0076

Tumor Conc. Plasma Conc. (μM) (nmol/g of tissue) Tumor/Plasma Ratio SDC-SDC- SDC- Analyte TRAP- TRAP- TRAP- Target 0076 SN-38 0076 SN-38 0076SN-38 Time (h) 0.083 671 — 73.4 8.66 — 0.503 0.01 — 0.01 1 52.9 — 8.609.12 — 0.642 0.17 — 0.07 6 4.00 — 1.18 8.98 — 0.670 2.25 — 0.57 24 0.359— 0.0755 7.32 — 0.572 20.4 — 7.58 48 1.11 — 0.160 7.60 — 0.489 6.85 —3.06

Tissue Distribution Data for SDC-TRAP-0154

Tumor Conc. Plasma Conc. (μM) (nmol/g of tissue) Tumor/Plasma Ratio SDC-SDC- SDC- SDC- SDC- SDC- Analyte TRAP- TRAP- TRAP- TRAP- TRAP- TRAP-Target 0154 0179 SN-38 0154 0179 SN-38 0154 0179 SN-38 Time (h) 0.083928 84.3 34.5 11.8 0.350 0.241 0.01 0.004 0.007 1 251 14.6 4.34 14.10.732 0.463 0.06 0.05 0.11 6 5.08 1.50 1.12 9.46 0.656 0.293 1.86 0.440.26 24 0.198 0.0428 0.0198 2.35 0.115 0.0562 11.9 2.68 2.84 48 0.02180.00344 BQL 1.88 0.0921 0.0465 86.0 26.8 —

Example 28: SDC-TRAP Comprising Fulvestrant

SDC-TRAP-0148

(7R,8R,9S,13S,14S,17S)-17-hydroxy-13-methyl-7-(9-((4,4,5,5,5-pentafluoropentyl)sulfinyl)nonyl)-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-yl4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazine-1-carboxylate

¹⁻H NMR (400 MHz, DMSO-d₆) δ 11.94 (s, 1H), 9.61 (s, 1H), 9.42 (s, 1H),7.30 (dd, J=25.2, 8.6 Hz, 3H), 7.18-7.11 (m, 2H), 6.88-6.75 (m, 3H),6.26 (s, 1H), 4.51 (dd, J=4.6, 2.5 Hz, 1H), 3.53 (d, J=16.6 Hz, 5H),2.97 (p, J=6.9 Hz, 1H), 2.91-2.58 (m, 8H), 2.43-2.22 (m, 6H), 2.04-1.77(m, 7H), 1.66-1.44 (m, 4H), 1.42-1.13 (m, 18H), 0.92 (dd, J=22.4, 7.1Hz, 6H), 0.67 (s, 3H); ESMS calculated for C₅₅H₇₂F₅N₅O₇S: 1041.51;Found: 1042.9 (M+H)⁺.

Example 29: SDC-TRAP Comprising Topotecan

SDC-TRAP-0159

10-((dimethylamino)methyl)-4-ethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl-1-(4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-(ethylcarbamoyl)-4H-1,2,4-triazol-4-yl)phenoxy)benzoyl)piperidine-4-carboxylate

ESMS calculated (C₅₆H₅₆N₈O₁₁): 1016.4; found: 1017.6 (M+H).

Example 30: SDC-TRAPs Comprising VDAs (Vascular Disrupting Agents)2-Methoxy-5-(5-(3,4,5-trimethoxyphenyl)isoxazol-4-yl)phenyl-4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazine-1-carboxylate

To a solution of 2-methoxy-5-(5-(3,4,5-trimethoxyphenyl)isoxazol-4-yl)phenol (0.1 g, 0.28 mmol) in THF (4 mL) was added 4-nitrophenylchloroformate (0.07 g, 0.35 mmol) and DIPEA (0.1 mL, 0.57 mmol). Thereaction was stirred at room temperature for 30 min before adding asolution of 4-(5-hydroxy-4-(4-(piperazin-1-ylmethyl)phenyl)-4H-1,2,4-triazol-3-yl)-6-isopropylbenzene-1,3-diol (0.13g, 0.31 mmol) and DIPEA (0.1 mL, 0.57 mmol) in DMF (2 mL). Afterstirring at room temperature for 30 min, the reaction was diluted withH₂O (10 mL), extracted with EtOAc (10 mL×3), and the combined organicphase was dried over Na₂SO₄ and concentrated. Column chromatography gaveSDC-TRAP-0098 (0.13 g, 59%).

¹H NMR (400 MHz, Methanol-d₄) δ 8.52 (s, 1H), 7.52-7.44 (m, 2H), 7.29(td, J=8.3, 2.0 Hz, 3H), 7.19-7.09 (m, 2H), 6.92 (s, 2H), 6.74 (s, 1H),6.29 (s, 1H), 3.85 (s, 3H), 3.80 (s, 3H), 3.73 (s, 6H) 3.68 (s, 2H),3.62 (s, 2H), 3.53 (s, 2H), 3.03 (p, J=6.9 Hz, 1H), 2.52 (t, J=4.7 Hz,4H), 0.92 (d, J=6.9 Hz, 6H); ESMS calculated (C₄₂H₄₄N₆O₁₀): 792.3;found: 793.2 (M+H).

SDC-TRAP-0099

2-methoxy-5-(5-(3,4,5-trimethoxyphenyl)isoxazol-4-yl)phenyl-4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)phenyl)piperazine-1-carboxylate

¹H NMR (400 MHz, DMSO-d₆) δ 11.86 (s, 1H), 9.60 (s, 1H), 9.45 (s, 1H),8.87 (s, 1H), 7.33 (dd, J=8.5, 2.2 Hz, 1H), 7.27 (d, J=2.2 Hz, 1H), 7.20(d, J=8.6 Hz, 1H), 7.05 (d, J=9.0 Hz, 2H), 6.96 (d, J=9.0 Hz, 2H), 6.88(s, 2H), 6.79 (s, 1H), 6.26 (s, 1H), 3.79 (s, 3H), 3.70 (d, J=1.1 Hz,10H), 3.53 (s, 2H), 3.23-3.14 (m, 5H), 2.98 (p, J=6.8 Hz, 1H), 0.97 (d,J=6.8 Hz, 6H); ESMS calculated (C₄₁H₄₂N₆O₁₀): 778.3; found: 779.2 (M+H).

SDC-TRAP-0158

5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4-(4-(4-((1-((2-methoxy-5-(5-(3,4,5-trimethoxyphenyl)isoxazol-4-yl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)phenoxy)phenyl)-4H-1,2,4-triazole-3-carboxamideESMS calculated (C₅₅H₅₃N₇O₁₁): 987.4; found: 988.3 (M+H).

SDC-TRAP-0085

(Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenyl4-(4-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)benzyl)piperazine-1-carboxylate

A mixture of4-(5-hydroxy-4-(4-(piperazin-1-ylmethyl)phenyl)-4H-1,2,4-triazol-3-yl)-6-isopropylbenzene-1,3-diol(a, 0.1 mmol), (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenyl(4-nitrophenyl) carbonate (b, 0.1 mmol) and TEA (0.2 mmol) in DMF (2 mL)was stirred at room temperature for 2 days. The mixture was diluted withwater (50 mL) and extracted with EtOAc. The organic layers werecombined, concentrated and purified by column to give SDC-TRAP-0085 as awhite solid (13 mg, 0.02 mmol).

¹H NMR (400 MHz, Chloroform-d) δ 10.78 (s, 1H), 9.76 (s, 1H), 7.52 (d,J=8.0 Hz, 2H), 7.32 (d, J=8.1 Hz, 2H), 7.15-7.04 (m, 2H), 6.83 (d, J=8.5Hz, 1H), 6.56-6.38 (m, 6H), 6.35 (s, 1H), 3.82 (d, J=10.9 Hz, 6H), 3.71(s, 9H), 3.57 (d, J=16.1 Hz, 4H), 2.53 (s, 4H), 0.70 (d, J=6.8 Hz, 6H).ppm; ESMS calculated for C₄₁H₄₅N₅O₉: 751.3; found: 752.2 (M+H⁺).

SDC-TRAP-0025

1-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)-3-(5-fluoro-2-oxo-1,2-dihydropyrimidin-4-yl)urea

To a solution of 5-fluorocytosine (0.14 g, 1.1 mmol) in pyridine (4 mL)was added 4-nitrophenyl chloroformate (0.22 g, 1.1 mmol). The reactionwas heated in a microwave at 90° C. for 30 min. To the resultingsolution was added4-(5-hydroxy-4-(1-(2-hydroxyethyl)-1H-indol-5-yl)-4H-1,2,4-triazol-3-yl)-6-isopropylbenzene-1,3-diol(0.15 g, 0.38 mmol). The reaction was heated in microwave at 100° C. for1 hr. The solution was concentrated and column chromatography gaveSDC-TRAP-0025 (0.07 g, 34%).

¹H NMR (400 MHz, DMSO-d₆) δ 11.86 (s, 1H), 9.52 (s, 1H), 9.46 (d, J=4.8Hz, 1H), 8.10-7.82 (m, 2H), 7.59-7.39 (m, 3H), 6.95 (t, J=7.7 Hz, 1H),6.73 (d, J=9.6 Hz, 1H), 6.44 (dd, J=16.8, 3.3 Hz, 1H), 6.22 (s, 1H),4.31 (dt, J=12.6, 6.4 Hz, 2H), 3.57-3.48 (m, 2H), 2.90 (h, J=7.1 Hz,1H), 0.84 (t, J=7.8 Hz, 6H); ESMS calculated (C₂₆H₂₅FN₈O₅): 548.2;found: 549.1 (M+H).

in vitro activity was determined for these compounds using the HER2degradation assay set forth herein:

HER2 degradation STA# IC₅₀ (nM) SDC-TRAP-0148 3037 SDC-TRAP-0159 >1000SDC-TRAP-0098 232 SDC-TRAP-0099 677 SDC-TRAP-0158 >5000 SDC-TRAP-0085889 SDC-TRAP-0025 403

Mouse Plasma Stability Data

Compound ID % Remaining (1 h) SDC-TRAP-0098 96.0% SDC-TRAP-0099 95.2%SDC-TRAP-0158 92.7%

Tissue Distribution Data for SDC-TRAP-0098

Tumor Conc. Tumor/Plasma Plasma Conc. (μM) (nmol/g of tissue) Ratio SDC-SDC- SDC- SDC- SDC- SDC- SDC- SDC- Analyte TRAP- TRAP- TRAP- TRAP- TRAP-TRAP- TRAP- TRAP- Target .8 0052 0001 0098 0052 0001 0098 0052 0001 Time(h) 0.083 481 0.0833 0.700 5.02 0.0175 0.0360 0.01 0.21 0.05 1 7.480.437 0.250 4.62 0.111 0.161 0.62 0.25 0.65 6 0.387 0.131 0.0122 3.180.292 0.117 8.22 2.23 9.64 24 0.00306 0.0375 BQL 0.920 0.611 0.0614 30016.3 — 48 BQL 0.0125 BQL 0.182 0.770 0.0211 — 61.8 —

Example 31: SDC-TRAP-02325-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(morpholinomethyl)phenyl)-N-(5-sulfamoylpentyl)-4H-1,2,4-triazole-3-carboxamide

The synthesis of SDC-TRAP-0232 is outlined in the following scheme. Thefinal amide coupling was performed using boric acid as the catalyst inreflux dioxane. The synthesis of INT-2 is described elsewhere inliterature.

¹H NMR (400 MHz, DMSO-d₆) δ 8.93 (t, J=6 Hz, 1H), 7.39 (d, J=8 Hz, 2H),7.30 (d, J=8 Hz, 2H), 6.71 (bs, 1H), 6.53 (s, 1H), 6.28 (s, 1H), 3.59(bs, 4H), 3.50 (s, 2H), 3.31 (bs, 1H), 3.23-3.11 (m, 2H), 2.94-2.87 (m,2H), 2.38 (bs, 4H), 1.67-1.61 (m, 2H), 1.47-1.36 (m, 2H), 1.36-1.30 (m,2H), 0.78 (d, J=7.2 Hz, 6H). ESMS cacld (C28H38N6O6S): 586.26; found:587.2 (M+H).

Example 32: SDC-TRAP-233

SDC-TRAP-0233

N-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide

SDC-TRAP-0233 was synthesized from the corresponding HSP90 inhibitorusing standard amide coupling conditions.

¹H NMR (400 MHz, DMSO-d₆) δ 11.87 (s, 1H), 9.54 (s, 1H), 9.46 (d, J=4.8Hz, 1H), 7.94-7.93 (m, 1H), 7.47-7.36 (m, 3H), 6.95-6.92 (m, 1H), 6.77(s, 1H), 6.44-6.37 (m, 3H), 6.22 (s, 1H), 4.32-4.10 (m, 4H), 3.37-3.35(m, 2H), 3.10-3.06 (m, 1H), 2.95-2.88 (m, 1H), 2.84-2.79 (m, 1H), 2.58(d, J=12.0 Hz, 1H), 2.02 (t, J=8.0 Hz, 2H), 1.60-1.26 (m, 6H), 0.86 (t,J=7.8 Hz, 6H).

ESMS cacld (C31H37N7O5S): 619.2; found: 620.2 (M+H).

Example 33: SDC-TRAP-234

SDC-TRAP-0234

N-(2-(5-(3-(2,4-dihydroxy-5-isopropylphenyl)-5-hydroxy-4H-1,2,4-triazol-4-yl)-1H-indol-1-yl)ethyl)-6-(5-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamide

SDC-TRAP-0234 was synthesized starting from the corresponding HSP90inhibitor with the coupling of a Boc protected aminohexanoic acid.Subsequent deprotection followed by coupling of biotin using standardcoupling conditions afforded the desired product.

¹H NMR (400 MHz, DMSO-d₆) δ 11.86 (s, 1H), 9.55 (s, 1H), 9.46 (s, 1H),7.93 (t, J=6.0 Hz, 1H), 7.74 (t, J=6.0 Hz, 1H), 7.46 (d, J=8.0 Hz, 1H),7.41 (d, J=4.0 Hz, 1H), 7.35 (d, J=4.0 Hz, 1H), 6.94 (dd, J=8.0, 4.0 Hz,1H), 6.76 (s, 1H), 6.43-6.41 (m, 2H), 6.36 (s, 1H), 6.22 (s, 1H),4.31-4.10 (m, 4H), 3.09-2.79 (m, 8H), 2.05-2.01 (m, 4H), 1.61-1.12 (m,12H), 0.86 (t, J=7.8 Hz, 6H). ESMS cacld (C37H48N8O6S): 732.34; found:733.3 (M+H).

Example 34: Identification and Use of SDC-TRAP for Prevention andTreatment of Chronic Bronchitis and Asthma

Chronic bronchitis is a chronic inflammation of the bronchi in thelungs. It is generally considered one of the two forms of chronicobstructive pulmonary disease (COPD), the other being emphysema. It isdefined clinically as a persistent cough that produces sputum (phlegm)and mucus, for at least three months per year in two consecutive years.

Asthma is an inflammatory disorder that causes the airways of the lungsto swell and narrow, leading to wheezing, shortness of breath, chesttightness, and coughing. Asthma can be chronic or be triggered byenvironmental triggers including, but not limited to, animal hair ordander, dust, changes in weather, exercise, mold, and pollen.

Drugs used for the treatment of chronic bronchitis, COPD, and asthmainclude, but are not limited to, smooth muscarinic acetylcholinereceptor inhibitors such as ipratropium bromide; anticholinergicbronchodilators such as tiotropium; long-acting β2-adrenergic receptoragonists such as salmeterol, formoterol, and albuterol;anti-inflammatory agents such as inhaled steroids, montelukast, aleukotriene receptor antagonist (LTRA), and roflumilast, a selective,long-acting inhibitor of the enzyme phosphodiesterase-4 (PDE-4);xanthines such as theophylline; and mucolytic agents such as bromhexineand acetylcysteine. In cases where chronic bronchitis is caused orexacerbated by bacterial infection, antibiotics can be used fortreatment.

Many of the agents used for the treatment of chronic bronchitis, COPD,and asthma work through receptors that are present throughout the body,thereby potentially causing undesirable side effects. Although many ofthe drugs are available for administration by inhalation, which canincrease delivery to the target site and reduce side effects, decreasedlung function in the disease population may result in improper dosingand reduced compliance.

Roflumilast(3-(cyclopropylmethoxy)-N-(3,5-dichloropyridin-4-yl)-4-(difluoromethoxy)benzamide),a selective, long-acting inhibitor of the enzyme phosphodiesterase-4(PDE-4), is formulated as a tablet for oral administration and isapproved for use in the treatment of chronic bronchitis and COPD.Roflumilast can be used as a binding moiety in combination with one ormore drugs to make an SDC-TRAP that can be used for the treatment ofchronic bronchitis, COPD, or asthma, such as those listed above andthroughout the application, to target other agents to the site ofinterest, i.e., the lungs, while permitting oral delivery.

A roflumilast-effector molecule SDC-TRAP can be formed, for example,using any known linker, such as those provided herein, with the desiredeffector molecule. The specific linker and conjugation method used willdepend, for example, on the chemical nature of the effector molecule.

Assays to determine the cytotoxicity of the roflumilast SDC-TRAPmolecule conjugate are performed using methods similar to those providedin Example 4. Cell viability assays are performed on non-transformedcells, preferably lung cells, to identify SDC-TRAPs with acceptabletoxicities, preferably compounds with toxicity that is not greater thaneither of the parent compounds.

Roflumilast SDC-TRAP molecules are also tested to confirm that theirefficacy is not inhibited by the formation of the complex. Assays totest PDE-4 activity are well known in the art and are commerciallyavailable (e.g., PerkinElmer LANCE® Ultra cAMP kit). The activity of theeffector molecule is tested using appropriate methods.

Methods to assess pharmacokinetic and pharmacodynamic properties of anagent are well known in the art. Tissue distribution studies areperformed to assess distribution of the conjugate as compared todistribution of each roflumilast and the effector molecule. An increaseaccumulation of the roflumilast SDC-TRAP molecules in the lung ascompared to the unconjugated effector molecule is observed. Such assaysare performed using orally delivered SDC-TRAPs of active agents that maytypically be administered by inhalation. Roflumilast SDC-TRAP moleculesare also identified for having longer serum stability.

Having identified roflumilast SDC-TRAP molecules with the desiredactivity, cytotoxicity, pharmacokinetic properties, and improvedpulmonary delivery, the SDC-TRAPs are tested for their efficacy of anappropriate animal model of chronic bronchitis, COPD, and/or asthma.Animal models of chronic bronchitis, COPD, and asthma are well known inthe art. The activity of the conjugate is compared to the activity ofeach roflumilast and the effector molecule alone. Roflumilast SDC-TRAPmolecules having one or more improved properties as compared to eitherof the parent molecules are further characterized in other animalsystems and humans.

The SDC-TRAPs are found to have one or more improved properties in thetreatment of humans including, but not limited to, decreased toxicity,improved dosing schedule, or improved efficacy.

Example 32: Identification and Use of SDC-TRAP for Prevention andTreatment of Skin Cancers and Actinic Keratosis

Skin cancers (neoplasms) are named after the type of skin cell fromwhich they arise. Skin cancers include basal cell carcinoma, squamouscell carcinoma, malignant melanomas, and Bowen's disease. Actinickeratosis can be, but is not always, a precursor to squamous cellcarcinoma.

Drugs used for the treatment of skin cancer are selected based on thetype and severity of the skin cancer. Superficial, non-melanoma skincancers can be treated with topical agents, either alone or incombination with surgery or other therapeutic interventions. Such agentsinclude, but are not limited to, retinoids, 5-fluorouracil, diclofenac,ingenol mebutate, and imiquimod. Topical delivery permits administrationof the chemotherapeutic agent directly to the site of the tumor or skinlesion. However, the delivery of active agents into the skin can bechallenging. Moreover, many topical therapeutic agents can be irritatingto the skin, resulting in scar formation, further inhibiting thedelivery of the active agent to the site.

Imiquimod3-(2-methylpropyl)-3,5,8-triazatricyclo[7.4.0.02,6]trideca-1(9),2(6),4,7,10,12-hexaen-7-amine)is a patient-applied cream used to treat certain diseases of the skin,including skin cancers (basal cell carcinoma, Bowen's disease,superficial squamous cell carcinoma, some superficial malignantmelanomas, and actinic keratosis) as well as genital warts (condylomataacuminata). Imiquimod and its analogs activate the immune system byactivating immune cells through the toll-like receptor 7 (TLR7),commonly involved in pathogen recognition. Imiquimod can be used incombination with one or more drugs used for the treatment of skindiseases to make an SDC-TRAP molecule.

An imiquimod SDC-TRAP molecule can be formed, for example, using anyknown linker, such as those provided herein, with the desired effectormolecule. The specific linker and conjugation method used will depend,for example, on the chemical nature of the effector molecule.

Assays to determine the cytotoxicity of the imiquimod SDC-TRAP moleculesare performed using methods similar to those provided in Example 4. Cellviability assays are performed on non-transformed cells, preferably skincells, to identify SDC-TRAPs with acceptable toxicities, preferablycompounds with toxicity that is not greater than either of the parentcompounds. Cytotoxicity and skin irritation assays are also performed,for example, on pig skin, which is frequently used as a model for humanskin in toxicity/irritation assays, using routine methods.

Imiquimod SDC-TRAP molecules are also tested to confirm that theirefficacy is not inhibited by the formation of the conjugate. A number ofskin cancer cell lines are well known in the art. Dose response curvesare generated to demonstrate the efficacy of imiquimod SDC-TRAPmolecules in killing cancer cells. Preferably, the imiquimod SDC-TRAPmolecules are more effective at killing skin cancer cells than imiquimodor the effector molecule alone.

Methods to assess pharmacokinetic and pharmacodynamic properties of anagent are well known in the art. As noted above, pig skin is frequentlyused as a model for human skin, both in toxicity/irritation assays, butalso in assaying uptake and delivery of agents into skin layers andcells. Topical formulations of imiquimod, the effector molecule, andimiquimod SDC-TRAP molecules are assayed for uptake, transport throughthe skin, and persistence in the skin using routine methods.

Having identified a imiquimod SDC-TRAP molecule with the desiredactivity, cytotoxicity, pharmacokinetic properties, and improved tissuedelivery, the SDC-TRAPs are tested for their efficacy in an appropriateanimal model of skin cancer. A animal models of skin cancer are wellknown in the art. For example, xenograph tumor models using squamouscell carcinoma, basal cell carcinoma, or melanoma cell lines are usedwith subcutaneously implanted tumors. Topical formulations of imiquimod,the effector molecule, and imiquimod SDC-TRAP molecules are applied. Theactivity of the conjugate is compared to the activity of each imiquimodand the effector molecule alone. Imiquimod SDC-TRAP molecules having oneor more improved properties as compared to either of the parentmolecules are further characterized in other animal systems and humans.

The SDC-TRAPs are found to have one or more improved properties in thetreatment of humans including, but not limited to, decreased toxicity,improved dosing schedule, or alternate route of administration.

Example 33: Determining the Permeability of SDC-TRAP Molecules

In order to test the ability SDC-TRAP molecules of the invention toenter cells, an artificial membrane permeability assay (“PAMPA”) wasused. PAMPAs are useful tool for predicting in vivo drug permeabilityfor drugs that enter cells by passive transport mechanisms. LC/MS wasused in conjunction with PAMPA assays to determine the ability of theSDC-TRAP molecules of the invention to permeate cells.

Pre-coated PAMPA plates were warmed to room temperature for at least 30minutes prior to adding assay components.

Stock solutions were prepared with the SDC-TRAP molecules to be tested.In order to make a working solution, either 50 μL of 100 μM Stock inDMSO+950 μL of PBS or 50 μL of 200 μM stock was added to 96 deep wellplate, resulting in a 5 μM final concentration or a 10 μM finalconcentration, respectively. 300 μL of the working solution containingeach compound to be tested was added to the appropriate well of a donorPAMPA plate. 200 μL of PBS was added into the corresponding wells of anacceptor PAMPA plates.

The acceptor plate was lowered onto the donor plate and allowed toincubate for five hours. After five hours, a 50 μL aliquot was removedfrom each well of each plate and added into a new 96 deep-well plate.

100 μL of methanol containing an internal standard was added to eachaliquot and analyzed by LC/MS. The internal standard was 150 ng/mlSDC-TRAP-0002.

In order to calculate the permeability for each SDC-TRAP molecule andthe control molecules, the following formula was used:

Permeability  (in  unit  of  cm/s):$P_{e} = \frac{- {\ln \left\lbrack {1 - {{C_{A}(t)}\text{/}C_{equilibrium}}} \right\rbrack}}{A*\left( {{1\text{/}V_{D}} + {1\text{/}V_{A}}} \right)*t}$$C_{equilibrium} = \frac{{{C_{D}(t)}*V_{D}} + {{C_{A}(t)}*V_{A}}}{V_{D} + V_{A}}$${{Mass}\mspace{14mu} {Retention}\text{:}\mspace{14mu} R} = \frac{1 - \left\lbrack {{{C_{D}(t)}*V_{D}} + {{C_{A}(t)}*V_{A}}} \right\rbrack}{C_{0}*V_{D}}$

C₀=initial compound concentration in donor well (mM)

C_(D) (t)=compound concentration in donor well at time t. (mM)

C_(A) (t)=compound concentration in acceptor well at time t. (mM)

V_(D)=donor well volume=0.3 mL

V_(A)=acceptor well volume=0.2 mL

A=filter area=0.3 cm²

t=incubation time=18000 s (5 h)

For the data presented in the table below, peak area was used in placeof concentration in the formula above.

Mass Permeability Retention SDC-TRAP-# (cm/s) (10⁻⁶ cm/s) (%)SDC-TRAP-0018 2.68E−08 0.0268 14.7 SDC-TRAP-0048 2.83E−08 0.0283 10.8SDC-TRAP-0049 1.24E−08 0.0124 14.1 SDC-TRAP-0052 7.69E−09 0.00769 7.02SDC-TRAP-0062 2.50E−08 0.025 18.0 SDC-TRAP-0193 8.59E−09 0.00859 10.2SDC-TRAP-0195 0.00E+00 0 27.1 SDC-TRAP-0196 0.00E+00 0 22.3SDC-TRAP-0210 0.00E+00 0 34.8 SDC-TRAP-0232 6.89E−09 0.00689 21.0SDC-TRAP-0233 2.10E−08 0.021 10.9 SDC-TRAP-0234 1.23E−08 0.0123 9.56Doxorubicin 3.30E−09 0.0033 21.0 Docetaxel 5.00E−08 0.05 17.6 SN-386.43E−07 0.643 38.2 Lenalidomide 6.20E−08 0.062 26.0 Furosemide 1.47E−080.0147 7.53 Caffeine 1.17E−05 11.7 20.8

The same protocol was used to test the permeability of the SDC-TRAPmolecules identified in the table below.

Mass Permeability Retention SDC-TRAP-# (cm/s) (10⁻⁶ cm/s) (%)SDC-TRAP-0029 6.46E−09 0.00646 84.0 SDC-TRAP-0046 1.22E−08 0.0122 88.1SDC-TRAP-0063   0E+00 0 18.7 SDC-TRAP-0064   0E+00 0 48.4 SDC-TRAP-0154  0E+00 0 10.3 SDC-TRAP-0200   0E+00 0 10.6 SDC-TRAP-0205   0E+00 0 10.9SDC-TRAP-0208   0E+00 0 25.0 SDC-TRAP-0210 8.99E−09 0.00899 72.2 SN-381.87E−06 1.87 46.6 Furosemide 2.50E−08 0.025 2.63 Caffeine 1.43E−05 14.3−0.11

All publications, patent applications, patents, and other documentscited herein are incorporated by reference in their entirety. In case ofconflict, the present specification, including definitions, willcontrol.

The specification should be understood as disclosing and encompassingall possible permutations and combinations of the described aspects,embodiments, and examples unless the context indicates otherwise. One ofordinary skill in the art will appreciate that the invention can bepracticed by other than the summarized and described aspect,embodiments, and examples, which are presented for purposes ofillustration, and that the invention is limited only by the followingclaims.

We claim:
 1. A method for treating a subject having a disease ordisorder comprising administering a therapeutically effective amount ofat least one binding moiety-drug conjugate (SDC-TRAP) to the subject,thereby treating the disease or disorder, wherein the SDC-TRAPAcomprises a binding moiety and an effector moiety, wherein the bindingmoiety binds to Hsp90 and wherein the effector moiety is a cytotoxicagent selected from the group consisting of flavopiridol, lapatinib,axitinib, vemurafenib, imatinib, staurosporine, panobinostat,carfilzomib, INCB024360 and methyl tryptophan or (a) fragment(s)thereof, and wherein the binding moiety and the effector moiety arecovalently attached.
 2. The method of claim 1, wherein the disease ordisorder is selected from the group consisting of: cancer, actinickeratosis, chronic bronchitis and asthma.
 3. The method of claim 2,wherein the cancer is lung cancer, colon cancer, breast cancer, skincancer, or bladder cancer.